Method for preparing water-soluble polymer derivatives bearing a terminal carboxylic acid

ABSTRACT

A method is provided for preparing water-soluble polymer derivatives bearing a terminal carboxylic acid or ester thereof. The method involves the hydrolyzing an ortho ester of a water-soluble polymer so as provide the corresponding acid. In addition, the invention provides water-soluble polymers bearing a terminal carboxylic acid or ester thereof, intermediates and reagents useful in carrying out the method, as well as gels, pharmaceutical formulations, conjugates related to the described water-soluble polymer derivatives.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/659,735, filed Sep. 9, 2003, now U.S. Pat. No. 7,569,214, whichapplication claims the benefit of priority to U.S. Provisional PatentApplication Ser. No. 60/409,348, filed Sep. 9, 2002, the disclosures ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to novel methods for preparingpolymer derivatives that comprise a terminal carboxylic acid or esterthereof. In addition, the invention relates to polymers, conjugates ofthe polymers, conjugation methods, and intermediates as well as methodsfor preparing the intermediates. Furthermore, the invention relates topharmaceutical preparations, synthetic methods, and the like.

BACKGROUND OF THE INVENTION

Conjugating a water-soluble polymer such as poly(ethylene glycol) (or“PEG”) to a biologically active agent results in a polymer-active agentconjugate that often has advantageous properties over the corresponding“unconjugated” version of the active agent. Among other advantages,conjugated forms of active agents have increased half-lives and are lessimmunogenic. When PEG is used to form a polymer-active agent conjugate,the conjugated active agent is conventionally referred to as“PEGylated.” Commercially available PEGylated preparations includePEGASYS® PEGylated interferon alpha-2a (Hoffmann-La Roche, Nutley,N.J.), PEG-INTRON® PEGylated interferon alpha-2b (Schering Corp.,Kennilworth, N.J.), NEULASTA™ PEG-filgrastim (Amgen Inc., Thousand Oaks,Calif.) and SOMAVERT® pegvisomant (Pfizer, New York, N.Y.). Thecommercial success of these preparations attests to the value ofPEGylation technology.

Polymers bearing a terminal carboxylic acid are useful, either directlyor indirectly, in conjugation reactions with active agents and othersubstances. For example, carboxylic acids can be reacted directly withan amino or hydroxyl group of an active agent, thereby forming aconjugate. Indirectly, polymers bearing a terminal carboxylic acid(which acts as a reactive electrophilic group) can serve as a convenientstarting material for preparing other polymer derivatives bearingfunctional groups other than carboxylic acids. Polymers bearing afunctional group other than a carboxylic acid can then form conjugateswith active agents bearing a suitable reactive group.

Methods for preparing certain water-soluble polymers bearing a terminalcarboxylic acid have been described. For example U.S. Pat. No. 5,681,567describes reacting a poly(alkylene oxide) with a tertiary-alkylhaloacetate to thereby form a tertiary-alkyl ester of a poly(alkyleneoxide) carboxylic acid. Schematically, the reaction using atertiary-alkyl chloroacetate can be represented as follows:

wherein each R is alkyl. Subsequent reaction of the ester with an acidremoves the tertiary alkyl moiety, which yields the corresponding aceticacid. This method, however, only results in polymers bearing a terminalacetic acid moiety. Polymer derivatives synthesized to terminate in anacetic acid moiety are sometimes referred to as “carboxymethylated”polymers.

Polymer derivatives bearing a terminal acetic acid can be furtherreacted to form polymer derivates bearing other reactive moieties. Forexample, a succinimidyl ester of carboxymethyl PEG can be formed. Thissuccinimidyl ester, however, is so reactive that it hydrolyzes almostimmediately in aqueous solution. Thus, the practical utility of PEGderivatives bearing a terminal acetic acid moiety can be low given theoverly reactive nature of these derivatives.

Another method for preparing certain water-soluble polymers bearing aterminal carboxylic acid derivative is described in U.S. Pat. No.5,523,479. In this approach, a moiety having a molecular weight of from32 to 6000 and having from one to 6 hydroxyl groups is reacted with atertiary alkyl ester of a beta-unsaturated carboxylic acid to yield aproduct having a terminal ester. Schematically, the reaction in can berepresented as follows (the moiety is presented as having a singlehydroxyl group and the tertiary alkyl ester of a beta-unsaturatedcarboxylic acid is represented by tertiary alkyl ester of acrylic acid)

wherein R is alkyl. A subsequent hydrolytic step transforms the esterinto the corresponding propanoic acid.

While providing polymer derivatives that lack a reactive acetic acidmoiety, this method suffers from other drawbacks. First, the methodinherently provides for only propanoic acid derivates. In addition, thebest reported conversion of the hydroxyl group to the ester is less than85%. Finally, only moieties having a molecular weight between 32 and6000 are described in connection with carrying out the method. Thereremains a need, however, to provide a method that can prepare acidsother than propanoic acid derivatives, result in conversion to an esterand/or acid of greater than 85%, and be used with moieties having amolecular weight outside of the range of 32 to 6000.

U.S. Pat. No. 5,672,662, discloses PEG derivatives having a terminalpropanoic acid or butanoic acid moiety that can be used to prepareactive esters suitable for conjugation to proteins or other moleculesbearing amino groups. The active esters described in U.S. Pat. No.5,672,662 exhibit greater stability in solution than active esters ofcarboxymethylated PEG, and are thus better suited for conjugation tobiologically active molecules. The method described for preparing thesePEG derivatives having a terminal propanoic or butanoic moiety, however,involves numerous steps and only results in about 80% substitution intothe carboxylic acid moiety. As a consequence, the method described inU.S. Pat. No. 5,672,662 requires expensive and time-consumingpurification steps in order to provide a pharmaceutical grade product.

Thus, there remains a need in the art for improved methods for preparingpolymer derivatives bearing a terminal carboxylic acid. In addition,there continues to be a need to provide novel polymers bearing acarboxylic acid moiety that are useful for conjugation reactions andfurther functionalization. The present invention addresses these andother needs in the art by providing, inter alia, novel methods for theefficient preparation of polymer derivatives bearing a terminalcarboxylic acid.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of this invention to provide amethod for making a carboxylic acid of a water-soluble polymercomprising the steps of (a) reacting a water-soluble polymer segmenthaving at least one alkoxide ion or thiolate ion with an ortho estercomprised of a suitable leaving group to form an ortho ester of awater-soluble polymer; and (b) subjecting the ortho ester of awater-soluble polymer formed in step (a) to one or more hydrolysis stepsso as to provide the corresponding carboxylic acid of a water-solublepolymer.

It is another object of the invention to provide an ortho ester usefulin the method for making the carboxylic acid of the water-solublepolymer. Thus, this object of the invention comprises carrying out step(a) recited in the immediately preceding paragraph.

It is yet another object of the invention to provide a carboxylic acidof a water-soluble polymer prepared by a method described herein.

It is a further object of the invention to provide a carboxylic acid orester thereof of a water-soluble polymer.

It is still another object of the invention to provide an ortho ester ofa water-soluble polymer.

It is still yet another object of the invention to provide gels,conjugates, and pharmaceutical compositions comprising a polymerdescribed herein.

It is another object of the invention to provide methods for preparingeach of the gels, conjugates, and pharmaceutical compositions describedherein.

It is a further object of the invention to provide methods foradministering each of the gels, conjugates, and pharmaceuticalcompositions described herein, comprising the step of delivering thepreparation to a patient.

Additional objects, advantages and novel features of the invention willbe set forth in the description that follows, and in part, will becomeapparent to those skilled in the art upon the following, or may belearned by practice of the invention.

In one embodiment of the invention then, an ortho ester of awater-soluble polymer is provided. Among other uses, the ortho ester hasutility as an intermediate in the synthesis of a water-soluble polymerbearing a terminal carboxylic acid group. The ortho ester of thewater-soluble polymer preferably comprises the following structure:

wherein:

POLY is a water-soluble polymer segment;

(a) is either zero or one;

X, when present, is a spacer moiety;

(z) is an integer from 1 to 24;

R¹, in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl;

R², in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl; and

represents a residue of a ortho ester moiety.

In another embodiment, the present invention provides a method formaking an ortho ester of a water-soluble polymer. The method comprisesthe step of reacting, in the presence of a base, a water-soluble polymersegment having at least one hydroxyl or thiol group with an ortho estercomprised of a suitable leaving group. It is preferred that thewater-soluble polymer segment has at least one hydroxyl group and lacksany thiol groups.

Typically, although not necessarily, the ortho ester comprising asuitable leaving group is comprised of the following structure:

wherein:

is the suitable leaving group;

(z) is an integer from 1 to 24;

R¹, in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl;

R², in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl; and

represents a residue of a ortho ester moiety.

In a further embodiment of the invention, a method for making acarboxylic acid of a water-soluble polymer is provided. The methodcomprises the step of subjecting an ortho ester of a water-solublepolymer to one or more hydrolysis steps so as to provide thecorresponding carboxylic acid of a water-soluble polymer. Although asingle hydrolysis step can be performed, it is preferred that twosequential hydrolysis steps are performed. Exemplary double hydrolysissteps include an initial base hydrolysis step followed by a second basehydrolysis step and an initial acid hydrolysis step followed by a basehydrolysis step.

In another embodiment, the invention provides a carboxylic acid of awater-soluble polymer prepared by the method. In this regard, polymerssuch as those having the following structure can be prepared:

wherein:

POLY is a water-soluble polymer segment;

(a) is either zero or one;

X, when present, is a spacer moiety;

(z) is an integer from 1 to 24;

R¹, in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl; and

R², in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl.

The corresponding ester versions of these acids are also includedwherein the terminal carboxylic moiety “—C(O)OH” of the polymer isreplaced by “—C(O)OR³” wherein R³ is defined as an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl.

Exemplary polymers bearing a terminal carboxylic acid or ester thereofinclude those having the following structure:

wherein:

POLY is a water-soluble polymer segment;

X′ is a spacer moiety with the proviso that when the spacer moiety isonly one atom, the one atom is not O or S;

(z′) is an integer from 3 to 24;

R¹, in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl;

R², in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl; and

R³ is H or an organic radical selected from the group consisting ofalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, andsubstituted alkynyl. Thus, it is preferred that R³ is nonaromatic.

For any structure comprising a water-soluble polymer segment, anypolymer that is water-soluble can be used and the invention is notlimited in this regard. Preferred water-soluble polymer segments,however, are terminally end-capped on one terminus. In addition,water-soluble polymer segments having a mass average molecular mass ofless than about 100,000 Daltons are preferred.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic representation of one approach for preparing awater-soluble polymer bearing a terminal carboxylic acid according tothe invention.

DETAILED DESCRIPTION OF THE INVENTION

Overview and Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to the particularpolymers, synthetic techniques, active agents, and the like as such mayvary. It is also to be understood that the terminology used herein isfor describing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to a“polymer” includes a single polymer as well as two or more of the sameor different polymers, reference to a “conjugate” refers to a singleconjugate as well as two or more of the same or different conjugates,reference to an “excipient” includes a single excipient as well as twoor more of the same or different excipients, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are meant to encompass any water-soluble poly(ethylene oxide).Typically, PEGs for use in accordance with the invention comprise thefollowing structure “—O(CH₂CH₂O)_(m)—” where (m) is 2 to 4000. As usedherein, PEG also includes—depending upon whether or not the terminaloxygen(s) has been displaced—the following similar structures“—CH₂CH₂—O(CH₂CH₂O)_(m)—CH₂CH₂—” and “—(CH₂CH₂O)_(m)—” where (m) is 2 to4000. When the PEG further comprises a linker moiety (to be described ingreater detail below), the atoms comprising the linker, when covalentlyattached to a water-soluble polymer segment, do not result in theformation of an oxygen-oxygen bond (i.e., an “—O—O—” or peroxidelinkage). Throughout the specification and claims, it should beremembered that the term “PEG” includes structures having variousterminal or “end capping” groups and so forth. The term “PEG” also meansa polymer that contains a majority, that is to say, greater than 50%, of—CH₂CH₂O— monomeric subunits. With respect to specific forms, the PEGcan take any number of a variety of molecular weights, as well asstructures or geometries such as “branched,” “linear,” “forked,”“multifunctional,” and the like, to be described in greater detailbelow.

The terms “end-capped” or “terminally capped” are used interchangeablyherein to refer to a terminal or endpoint of a polymer that terminateswith an end-capping moiety. Typically, although not necessarily, theend-capping moiety comprises a hydroxy or C₁₋₂₀ alkoxy group. Thus,examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxyand benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. In addition, saturated, unsaturated, substituted and unsubstitutedforms of each of the foregoing are envisioned. Moreover, the end-cappinggroup can also be a silane. The end-capping group can alsoadvantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) to which thepolymer is coupled to of interest can be determined by using a suitabledetector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, calorimetric (e.g.,dyes), metal ions, radioactive moieties, and the like. Suitabledetectors include photometers, films, spectrometers, and the like.

“Non-naturally occurring” with respect to a polymer or water-solublepolymer segment means a polymer that in its entirety is not found innature. A non-naturally occurring polymer or water-soluble polymersegment may, however, contain one or more subunits or portions of asubunit that are naturally occurring, so long as the overall polymerstructure is not found in nature.

The term “water soluble” as in a “water-soluble polymer segment” and“water-soluble polymer” is any segment or polymer that is soluble inwater at room temperature. Typically, a water-soluble polymer or segmentwill transmit at least about 75%, more preferably at least about 95% oflight, transmitted by the same solution after filtering. On a weightbasis, a water-soluble polymer or segment thereof will preferably be atleast about 35% (by weight) soluble in water, more preferably at leastabout 50% (by weight) soluble in water, still more preferably about 70%(by weight) soluble in water, and still more preferably about 85% (byweight) soluble in water. It is most preferred, however, that thewater-soluble polymer or segment is about 95% (by weight) soluble inwater or completely soluble in water.

“Molecular mass” in the context of a water-soluble, non-naturallyoccurring polymer of the invention such as PEG, refers to the nominalaverage molecular mass of a polymer, typically determined by sizeexclusion chromatography, light scattering techniques, or intrinsicvelocity determination in 1,2,4-trichlorobenzene. The polymers of theinvention are typically polydisperse, possessing low polydispersityvalues of preferably less than about 1.2, more preferably less thanabout 1.15, still more preferably less than about 1.10, yet still morepreferably less than about 1.05, and most preferably less than about1.03.

“Thiol derivative,” in the context of a water-soluble polymer, means apolymer having at least one terminus that is a thiol group (—SH), athiolate (—S⁻) or a protected thiol, that is to say, a thiol group inits protected form. Typical thiol protecting groups include thioether,thioester, or disulfide. Exemplary protecting groups for thiols can befound in Greene et al., “PROTECTIVE GROUPS IN ORGANIC SYNTHESIS,” 3^(rd)Edition, John Wiley and Sons, Inc., New York, 1999.

As used herein, the term “carboxylic acid” as in a “carboxylic acid”derivative is a moiety having a

functional group [also represented as a “—COOH” or —C(O)OH]. Unless thecontext clearly dictates otherwise, the term carboxylic acid includesnot only the acid form, but corresponding esters and protected forms aswell. Reference is again made to Greene et al. supra with respectsuitable protecting groups for carboxylic acids.

The term “reactive” or “activated” when used in conjunction with aparticular functional group, refers to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “nonreactive” or “inert” group).

The terms “protected” or “protecting group” or “protective group” referto the presence of a moiety (i.e., the protecting group) that preventsor blocks reaction of a particular chemically reactive functional groupin a molecule under certain reaction conditions. The protecting groupwill vary depending upon the type of chemically reactive group beingprotected as well as the reaction conditions to be employed and thepresence of additional reactive or protecting groups in the molecule, ifany. Protecting groups known in the art can be found in Greene et al.,supra.

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof.

The term “spacer” or “spacer moiety” is used herein to refer to an atomor a collection of atoms optionally used to link interconnectingmoieties such as a terminus of a water-soluble polymer segment and anelectrophile. The spacer moieties of the invention may be hydrolyticallystable or may include a physiologically hydrolyzable or enzymaticallydegradable linkage.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includeethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl when three or more carbon atoms are referenced.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, iso-butyl, and tert-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃-C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl (e.g., 0-2substituted phenyl); substituted phenyl; and the like. “Substitutedaryl” is aryl having one or more non-interfering groups as asubstituent. For substitutions on a phenyl ring, the substituents may bein any orientation (i.e., ortho, meta, or para).

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy,benzyl, etc.), preferably C₁-C₇.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup of 1 to 15 atoms in length, containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group of 2 to 15 atoms in length, containing at least onetriple bond, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl,octynyl, decynyl, and so forth.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof. Heteroaryl rings mayalso be fused with one or more cyclic hydrocarbon, heterocyclic, aryl,or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom which is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from non-interfering substituents.

“Electrophile” refers to an ion or atom or collection of atoms, that maybe ionic, having an electrophilic center, i.e., a center that iselectron seeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or collection of atoms that maybe ionic having a nucleophilic center, i.e., a center that is seeking anelectrophilic center or with an electrophile.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively weak bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater will depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include, butare not limited to, carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, ortho esters, peptides andoligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

The terms “active agent” and “biologically active agent” are usedinterchangeably herein and are defined to include any agent, drug,compound, composition of matter or mixture that provides somepharmacologic, often beneficial, effect that can be demonstrated in-vivoor in vitro. This includes foods, food supplements, nutrients,nutriceuticals, drugs, vaccines, antibodies, vitamins, and otherbeneficial agents. As used herein, these terms further include anyphysiologically or pharmacologically active substance that produces alocalized or systemic effect in a patient.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a polymer-active agent conjugate present ina pharmaceutical preparation that is needed to provide a desired levelof active agent and/or conjugate in the bloodstream or in the targettissue. The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofthe pharmaceutical preparation, intended patient population, patientconsiderations, and the like, and can readily be determined by one ofordinary skill in the art, based upon the information provided hereinand available in the relevant literature.

“Multifunctional” in the context of a polymer of the invention means apolymer having 3 or more functional groups contained therein, where thefunctional groups may be the same or different. Multifunctional polymersof the invention will typically contain from about 3-100 functionalgroups, or from 3-50 functional groups, or from 3-25 functional groups,or from 3-15 functional groups, or from 3 to 10 functional groups, orwill contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within thepolymer. A “difunctional” polymer means a polymer having two functionalgroups contained therein, either the same (i.e., homodifunctional) ordifferent (i.e., heterodifunctional).

“Branched,” in reference to the geometry or overall structure of apolymer, refers to polymer having 2 or more polymer “arms.” A branchedpolymer may possess 2 polymer arms, 3 polymer arms, 4 polymer arms, 6polymer arms, 8 polymer arms or more. One particular type of highlybranched polymer is a dendritic polymer or dendrimer, which, for thepurposes of the invention, is considered to possess a structure distinctfrom that of a branched polymer.

A “dendrimer” or dendritic polymer is a globular, size monodispersepolymer in which all bonds emerge radially from a central focal point orcore with a regular branching pattern and with repeat units that eachcontribute a branch point. Dendrimers exhibit certain dendritic stateproperties such as core encapsulation, making them unique from othertypes of polymers.

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as provided herein, and includes both humans and animals.

“Optional” and “optionally” mean that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

As used herein, the “halo” designator (e.g., fluoro, chloro, iodo,bromo, and so forth) is generally used when the halogen is attached to amolecule, while the suffix “ide” (e.g., fluoride, chloride, iodide,bromide, and so forth) is used when the ionic form is used when thehalogen exists in its independent ionic form (e.g., such as when aleaving group leaves a molecule).

In the context of the present discussion, it should be recognized thatthe definition of a variable provided with respect to one structure orformula is applicable to the same variable repeated in a differentstructure, unless the context dictates otherwise. Thus, for example, thedefinition of “POLY,” “a spacer moiety,” “(z),” and so forth withrespect to an ortho ester of a water-soluble polymer is equallyapplicable to a water-soluble polymer bearing a carboxylic acid.

The Method

The present methods for preparing a carboxylic acid of a water-solublepolymer have several advantages. As shown herein, for example,water-soluble polymers bearing a terminal carboxylic acid moiety can beprovided in high purity. Although prior art approaches result inrelatively low purity, conversions of at least about 85%, morepreferably at least about 90%, still more preferably at least about 95%,and most preferably at least about 98% conversion of a precursormolecule into the corresponding carboxylic acid derivative are shownherein. Because water-soluble polymers bearing a terminal carboxylicacid moiety can now be provided in relatively high purity, expensive andtime consuming purification steps are reduced or eliminated entirely.

Another advantage of the current methods for preparing a carboxylic acidof water-soluble polymer is the ability to prepare a larger range ofstructurally diverse derivatives. Previously described methodsnecessarily resulted in, for example, certain acetic acid orcarboxymethylated derivatives (see U.S. Pat. No. 5,681,567), certainpropanoic acid derivatives (see U.S. Pat. Nos. 5,523,479 and 5,672,662),and certain butanoic acid derivatives (see U.S. Pat. No. 5,672,662).These previously described derivatives are necessarily limited tocertain structures since the methods used to create them rely on arelatively narrow palette of possible reagents suitable for use withthese methods. Advantageously, the present methods can be used with arelatively large number of reagents, thereby greatly expanding the rangeof possible structures.

A first method is thus provided to form an ortho ester of awater-soluble polymer, which serves as a useful intermediate in furthercarrying out a step for subsequent formation of a water-soluble polymerbearing a terminal carboxylic acid. Process A as shown in FIG. 1,depicts one approach for carrying out this first method. One approachfor the subsequent formation of a water-soluble polymer bearing aterminal carboxylic acid (Formula V) is shown as process B in FIG. 1.Provided only for assistance in better understanding a synthetic methodpresented herein, FIG. 1 in no way should be construed as limiting theinvention. The various formulae shown in FIG. 1 are described in moredetail below.

Initially, the method for forming an ortho ester of a water-solublepolymer comprises the step of reacting a water-soluble polymer segmenthaving at least one alkoxide ion or thiolate ion with an ortho estercomprised of a suitable leaving group (i.e., an ortho ester-containingmolecule comprised of a suitable leaving group). Conveniently, and withreference to FIG. 1, the water-soluble polymer segment having at leastone alkoxide ion or thiolate ion is prepared by combining awater-soluble polymer having at least one hydroxyl or thiol group(Formula II) in the presence of a suitable base. An ortho estercomprising a suitable leaving group (Formula I) is allowed to react witha water-soluble polymer having at least one hydroxyl or thiol group(Formula II) to form an ortho ester comprised of a suitable leavinggroup (Formula III).

The base used in this approach, however, must be one that will form analkoxide (i.e., R—O⁻) or thiolate (i.e., R—S⁻) of the water-solublepolymer having at least one hydroxyl or thiol group, respectively. Thus,for example, the base transforms POLY-(X)_(a)—OH into POLY-(X)_(a)—O⁻and POLY-(X)_(a)—SH into POLY-(X)_(a)—S⁻. It is further believed thatthe water-soluble polymer, now bearing an alkoxide or thiolate moiety,in turn reacts via a S_(N)2 reaction mechanism with the ortho esterhaving a suitable leaving group (Formula I). As will be recognized bythose of ordinary skill in the art, this approach corresponds toWilliamson ether synthesis, and the principles and techniques generallyused in a Williamson ether synthesis are applicable here as well.

Nonlimiting examples of bases suitable to form an alkoxide of an alcoholor a thiolate of a thiol-containing compound include sodium, sodiumhydroxide, potassium, potassium hydroxide, sodium hydride, potassiumhydride, sodium methoxide, potassium methoxide, sodium tert-butoxide,potassium tert-butoxide, sodium carbonate, and potassium carbonate.Preferred bases for use in accordance with this step, however, includethose selected from the group consisting of sodium, potassium, sodiumhydride, potassium hydride, sodium methoxide, potassium methoxide,sodium tert-butoxide, and potassium tert-butoxide.

In addition, the water-soluble polymer segment having at least onealkoxide ion or thiolate ion can conveniently be provided via apolymerization reaction, as will be discussed in more detail below. Inthis approach for providing the water-soluble polymer segment, it ispreferred that the water-soluble polymer segment has at least onealkoxide ion.

Generally, although not necessarily, an excess of the ortho estercomprising a suitable leaving group (Formula I) is allowed to react withthe water-soluble polymer bearing at least one alkoxide ion or thiolateion (Formula II). Typically, the amount of the ortho ester comprising asuitable leaving group (Formula I) represents at least a molarequivalent to the number available hydroxyl or thiol groups in thewater-soluble polymer having at least one hydroxyl or thiol group(Formula II). Heterofunctional polymer species (i.e., species bearingtwo or more different terminal functional groups) can be prepared byusing nonstoichiometric amounts of the ortho ester comprising a suitableleaving group (Formula I). That is, heterofunctional species are formedwhen the total number of moles of available hydroxyl or thiol groups onthe water-soluble polymer having at least one hydroxyl or thiol group(Formula II) exceeds the total number of moles of the ortho estercomprising a suitable leaving group (Formula I) added to the reaction.

The ortho ester of the water-soluble polymer (Formula III) can beprepared by other means and the invention is not limited simply toprocess A as depicted in FIG. 1. For example, an ortho ester comprisingat least one initiator site suitable for polymerization can be used togrow one or more water-soluble polymer segments. Using this approach, anortho ester comprising at least one initiator site (e.g., an alkoxidemoiety) and a reactive monomer (e.g., ethylene oxide) are combined andthe reaction is allowed to proceed until all of the reactive monomer isexhausted or the reaction is terminated by, for example, neutralizingthe reaction medium. The last reactive monomer, e.g., ethylene oxide,added to the growing chain may conveniently provide an alkoxide ion orthiolate ion for subsequent reaction with, for example, an ortho estercomprised of a suitable leaving group.

Specifically, the following steps can be followed in order to build thewater-soluble polymer segment directly onto an ortho ester comprising atleast one initiator site: (i) providing an ortho ester comprising atleast one active anionic site suitable for initiating polymerization;(ii) contacting the anionic site of the ortho ester with a reactivemonomer capable of polymerizing, to thereby initiate polymerization ofthe reactive monomer onto the ortho ester precursor; (iii) addingaddition reactive monomers to the ortho acid precursor to form one ormore polymer chain(s); (iv) allowing said contacting to continue until adesired length of the polymer chain(s) is reached; and (v) terminatingthe reaction to achieve an ortho ester of a water-soluble polymer.

Any reactive monomer can be used to “grow” the polymer chain(s) so longas the resulting polymer chain is water soluble. It is particularlypreferred, however, that the reactive monomer is ethylene oxide, therebyproviding poly(ethylene oxide) chain(s). Growth of the polymer chain(s),including the initial attachment of the reactive monomer to theinitiator site, can be effected through, for example, an alkoxide ion(i.e., R—O³¹-). These and other techniques are known to those ofordinary skill in the art and are referenced in, for example, Odian,Chap. 7, Principles of Polymerization, 3^(rd) Ed., McGraw-Hill, 1991.

Growth of the polymer chain(s) continues until the desired molecularweight is achieved. Thus, for example, neutralizing the reaction mediumhalts the growth of the polymer chain(s). In addition, adding a specificweight or amount of the reactive monomer and allowing the polymerizationto proceed until all reactive monomer is exhausted results in a polymerchain having a corresponding molecular weight. Once the polymer chain(s)are formed, an end-capping group can be added using conventionaltechniques. For example, an alkyl halide (e.g., methyl halide or methylp-toluenesulfonate) can be reacted with the exposed terminal (theterminal distal to the ortho ester functionality) of the polymer chain.

A related, although different, polymerization approach can also be usedto provide an ortho ester of a water-soluble polymer. In this relatedapproach, the polymerization is carried out to first form a polymerchain that can be subsequently transformed into the ortho esterderivative. Thus, for example, an alkoxy alcoholate salt such as sodium2-methoxy ethanolate (Na⁺:⁻OCH₂CH₂OCH₃) can initiate polymerization ofethylene oxide by essentially following the same procedure outlinedabove. Assuming that the final monomer added to the polymer chain leavesa reactive group such as an alkoxide (as in the case of ethylene oxide),the polymer chain can then be reacted with an ortho ester comprising asuitable leaving group. To the extent that the polymer chain does notleave a group (e.g., an alkoxide) suitable for direct attachment to anortho ester-containing reagent, additional modifications to the polymerchain can be made such that an ortho ester can be attached.

Use of an alkoxy alcoholate salt as an initiator of the polymer resultsin an ortho ester of a water-soluble polymer comprising a single orthoester functionality. Polymers comprising two ortho estersfunctionalities (a bifunctional polymer) can result when dialcoholatesalts (e.g. 2Na⁺:⁻OCH₂CH₂O⁻) are used in place of alkoxy alcoholatesalts. As described above, an optional capping step can also beperformed with this polymerization approach.

Returning to FIG. 1, the ortho ester of a water-soluble polymer (FormulaIII) can be converted into a water-soluble polymer bearing a terminalcarboxylic (Formula V). The conversion into the corresponding carboxylicacid derivative is advantageously accomplished efficiently and in highyield by performing one or more hydrolysis steps. Either acid-catalyzedhydrolysis or acid-catalyzed hydrolysis followed by base-promotedhydrolysis can provide the water-soluble polymer bearing a terminalcarboxylic acid (Formula V).

Although conversion into the carboxylic acid can be carried out in asingle hydrolysis step via acid-catalyzed hydrolysis, it is believedthat this single hydrolytic approach is inefficient in terms of time. Ithas been found that two hydrolysis steps, however, increases the speedfor converting the carboxylic acid from the corresponding ortho ester.

In a two hydrolysis step approach, a first hydrolysis step results inthe ortho ester functionality being transformed into an ester (FormulaIV). A second hydrolysis step, in turn, converts the ester (Formula IV)to the corresponding polymer bearing a terminal carboxylic acid (FormulaIV).

The first hydrolysis step should be acid-catalyzed hydrolysis. The orthoester functionality can be cleaved by mild aqueous acidic conditionssuch asp-toluenesulfonic acid (p-TsOH) and pyridine in water, and NaHSO₄and 1,2-dimethoxyethane (DME) in water at 0° C. for 20 minutes. See Justet al. (1983) Can. J. Chem. 61:712 and Corey et al. (1986) TetrahedronLett. 27:2199, respectively. Examples of other acids suitable for use inacid-catalyzed hydrolsis include, without limitation, hydrofluoric acid(HF), hydrochloric acid (HCl), hydrobromic acid (HBr), hydroiodic acid(HI), nitric acid (HNO₃), perchloric acid (HClO₄), sulfuric acid(H₂SO₄), acetic acid (CH₃CO₂H), carbonic acid H₂CO₃, phosphoric acid(H₃PO₄), oxalic acid (H₂C₂O₄), and formic acid (HCOOH).

The second hydrolysis step is typically a base-promoted hydrolysis step.The ester (Formula IV) of the first hydrolysis step is treated with abase. For base-promoted hydrolysis, the ortho ester functionality istreated with any aqueous base, such as lithium hydroxide (LiOH), sodiumhydroxide (NaOH), potassium hydroxide (KOH), rubidium hydroxide (RuOH),cesium hydroxide [Cs(OH)₂], strontium hydroxide [Sr(OH)₂], bariumhydroxide [Ba(OH)₂], ammonium hydroxide (NH₄OH), magnesium hydroxide[Mg(OH)₂], calcium hydroxide [Ca(OH)₂], sodium acetate (NaCH₃CO₂),potassium acetate (KCH₃CO₂), sodium carbonate (Na₂CO₃), potassiumcarbonate (K₂CO₃), lithium carbonate (Li₂CO₃), sodium phosphate(Na₃PO₄), potassium phosphate (K₃PO₄), sodium borate (Na₃BO₄), potassiumborate (Li₃PO₄), and so forth.

The first, and optional second hydrolysis steps can be carried out underincreased heat in order to increase the rate reaction.

The steps of the method take place in an appropriate solvent. One ofordinary skill in the art can determine whether any specific solvent isappropriate for any given reaction. Typically and in particular withrespect processes A and B as shown in FIG. 1, however, the solvent ispreferably a nonpolar solvent or a polar aprotic solvent. Nonlimitingexamples of nonpolar solvents include benzene, xylene, dioxane,tetrahydrofuran (THF), t-butyl alcohol and toluene. Particularlypreferred nonpolar solvents include toluene, xylene, dioxane,tetrahydrofuran, and t-butyl alcohol. Exemplary polar aprotic solventsinclude, but are not limited to, DMSO (dimethyl sulfoxide), HMPA(hexamethylphosphoramide), DMF (dimethylformamide), DMA(dimethylacetamide), NMP (N-methylpyrrolidinone).

With respect to hydrolysis, in particular, water is a preferred solvent,although aqueous mixtures of water with other solvents such as water andtetrahydrofuran, water and 1,2-dimethylethane, water and diglyme, aswell as other aqueous-containing solvents can be used.

Optionally, the method further comprises the step of recovering thecarboxylic acid of the water-soluble polymer. Art-known techniques canbe used to recover the polymer and include, for example, precipitatingthe polymer from solution. The precipitate can then be collected andoptionally filtered and/or dried. These and other techniques can be usedto isolate and recover the carboxylic acid of the water-soluble polymer.

In addition, the method optionally includes the step of furtherpurifying the carboxylic acid of the water-soluble polymer. Such apurifying step, however, is generally not required given the relativelyhigh degree of converting a precursor (e.g., ortho ester of awater-soluble polymer) into the corresponding carboxylic acid. In anyevent, art-known techniques such chromatography can be used to purifythe polymer.

The acids so produced according to the present method are substantiallypure and can be prepared in a one-pot approach. By pure, it is meantthat preferably greater than at least about 85%, more preferably greaterthan at least about 90%, still more preferably greater than at leastabout 95%, and most preferably greater than at least about 98% of thetotal amount (either by weight or molar basis) of the water-solublepolymer bearing a hydroxyl or thiol group is converted to water-solublepolymer bearing a terminal carboxylic acid.

The Ortho Ester of a Water-Soluble Polymer

As indicated above, the method requires use of an ortho ester comprisinga suitable leaving group. Structurally, the ortho ester comprises a“branching carbon atom.” This branching carbon atom is a carbon atomcovalently attached to three oxygen atoms, which, in turn, are typicallyeach covalently attached to, for example, an alkyl moiety. Given thetetravalent nature of carbon, the branching carbon atom also comprises afourth covalent attachment. In the case of the present ortho esters ofthe invention used to make alkanoic acids, the fourth covalentattachment of the branching carbon atom is to a substituted orunsubstituted carbon chain, such as an alkylene chain. Finally, asuitable leaving group is attached, either directly or though a spacermoiety, at the end of the carbon chain that is not attached to thebranching carbon atom.

An exemplary structure of an ortho ester comprising a suitable leavinggroup is provided below.

wherein:

is the suitable leaving group;

(z) is an integer from 1 to 24;

R¹, in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl;

R², in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl; and

represents a residue of a ortho ester moiety.

With respect Formula I, the suitable leaving group is any atom or groupof atoms that can leave the carbon atom to which it is attached.Specifically, a suitable leaving group is one that can be displaced byan approaching nucleophile. Those of ordinary skill in the art candetermine what atom or group of atoms can serve as a suitable leavinggroup. In addition, routine experimentation can identify whether anyspecific atom or group of atoms can serve as a suitable leaving group.For example, a proposed leaving group on a molecule comprising an orthoester can be tested by reacting the ortho ester with a water-solublepolymer segment having a hydroxyl group; the proposed leaving group is asuitable leaving group if detectable amounts of the corresponding orthoester of the water-soluble polymer are formed.

Preferred suitable leaving groups include those that are primary (e.g.,a primary halo), although leaving groups that are secondary may also beused. Examples of suitable leaving groups include halogens and sulfonateesters. Among the halogens, bromo, chloro, iodo, and fluoro arepreferred, with bromo and chloro being particularly preferredhalogen-type leaving groups. With respect to sulfonate esters,methanesulfonate, trifluoromethanesulfonate, trichloromethanesulfonate,2,2,2-trifluoroethanesulfonate, 2,2,2-trichloroethanesulfonate, andpara-toluenesulfonate are particularly preferred, although othersulfonate esters and similarly constituted leaving groups known to thoseof ordinary skill in the art can be used as well.

With respect to the specific ortho ester functionality associated withFormula I, any ortho ester functionality can be used and the inventionis not limited in this regard. An exemplary ortho ester functionality,however, is comprised of the following structure:

wherein each R⁴ is an organic radical independently selected from thegroup consisting of alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, and oneor more atoms that combine with another R⁴ or the remaining R⁴ moietiesto form a ringed structure.

The ortho ester functionality can be acyclic, i.e., lacking a ringedstructure. When acyclic, it is preferred that each R⁴ in theabove-defined structure is independently a C₁₋₆ alkyl (e.g., methyl,ethyl or propyl) or substituted C₁₋₆ alkyl. In addition, the ortho esterfunctionality can be in form of a “cyclic” or “ringed” structure. In thepresent context, the term “cyclic” will be understood to includemonocyclic, bicyclic, and polycyclic structures. In cyclic versions, theortho ester functionality is preferably in the form of a substituted orunsubstituted heterocyclic ring comprising from about 6 to about 14atoms. Preferred substituents for the heterocyclic ring include C₁₋₆alkyl, such as methyl or ethyl, or substituted C₁₋₆ alkyl. Examples ofsuch preferred cyclic structures include the following bridgedheterocyclic rings:

4-methyl-2,6,7-trioxabicyclo[2.2.2]octanyl, (“OBO ester”);

4-methyl-2,7,8-trioxabicyclo[3.2.1]octanyl, (“ABO ester”); and

2,8,9-trioxatricyclo[3.3.1.1^(3,7)]decanyl.

One of ordinary skill in the art can readily envision other cyclicstructures that comprise other ortho ester structures (both cyclic andacyclic).

As can be seen with respect to Formula I, the ortho ester comprising asuitable leaving group comprises a carbon chain of (z) carbons definedby the following structure:

wherein (z), each R¹ and each R² are as previously defined. Preferably,however, (z) is equal to one, two, three, four or five.

The carbon chain can be a simple, straight chain of carbon atoms.Simple, straight carbon chains are those in which each R¹ and R² isdefined as hydrogen. In addition, the carbon chain may comprise one ormore carbon-carbon double and/or triple bonds. Moreover, the carbonchain can be singly branched wherein one of R¹ and R² is defined as anatom or group of atoms other than hydrogen, such as alkyl, and all otherR¹ and R² variables are hydrogen. Multiple branching is also envisionedwherein multiple instances of R¹ and/or R² are defined as an atom orgroup of atoms other than hydrogen (e.g., alkyl). It is preferred,however, that branched species include only a single branching point. Inaddition, it is preferred that the single branch point occurs at thecarbon atom a to (or immediately adjacent to) the “branching carbonatom” in the ortho ester functionality.

Optionally, a spacer moiety can be located between the carbon chain andthe suitable leaving group. Exemplary ortho esters comprising such aspacer moiety comprise the following structure:

wherein:

X² is a spacer moiety and

(z), each R¹, each R², and

are as previously defined.

The optional spacer moiety (i.e., X²) in the ortho ester comprising asuitable leaving group is any atom or series of atoms separating thecarbon chain of (z) atoms from the leaving group. Depending on theactual atom or atoms that make up this spacer moiety, the spacer moietycan be hydrolytically stable or hydrolytically unstable. Whether anyspecific moiety is hydrolytically stable or unstable can be determinedby one of ordinary skill in the art or determined experimentally usingroutine experimentation. This optional spacer moiety X² can be selectedfrom the spacer moieties identified below with respect to X. In thoseinstances where X² appears within the same structure defined ascomprising an X or X¹, X² can be the same or different.

It should be stressed that although the ortho ester comprising asuitable leaving group comprises a carbon chain of (z) atoms, thepresence of the carbon chain is not necessary to provide a carboxylicacid. Consequently, when a water-soluble polymer bearing a terminalcarboxylic acid other than an alkanoic acid (e.g., a propanoic acid,butanoic acid, and so forth) is desired, the carbon chain is omitted andreplaced by, for example, a spacer moiety or other group of atoms.

The ortho ester having a suitable leaving group can be preparedsynthetically. For example, acyclic ortho esters can be prepared byobtaining an imino ester (also referred to as an “alkyl imidate”)through, for example, a Pinner reaction. In this approach, an iminoester is formed via the addition of anhydrous hydrogen chloride gas to amixture of a nitrile and an alcohol. Subsequent treatment of the iminoester with an alcohol yields the corresponding ortho ester. See, forexample, Voss et al. (1983) Helv. Chim. Acta. 66:2294.

Cyclic ortho esters can be prepared via conversion of ahydroxyalkyloxetane to the corresponding carboxylic ester, which,following rearrangement, yields a bridged ortho ester. These and otherapproaches for preparing cyclic ortho esters are described in theliterature. See, for example: Corey et al. (1983) Tetrahedron Lett.24(50):5571-5574; Wipf et al. (1999) Pure Appl. Chem. 71(3):415-421; andGreene et al. PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd ed., pp.437-441, John Wiley & Sons, New York, N.Y. (1999). The suitable leavinggroup is introduced in the ortho ester by including the leaving group inthe reactant prior to formation of the ortho ester or by adding itsubsequent to the formation of the ortho ester.

In some cases, the ortho ester comprising a suitable leaving group isavailable commercially. For example, one commercially available orthoester is trimethyl 4-bromoorthobutyrate available from Sigma-AldrichCorporation of St. Louis, Mo.

The Water-Soluble Polymer Having at Least One Hydroxyl or Thiol Group

Any water-soluble polymer having at least one hydroxyl or thiol group(to provide, for example, a water-soluble polymer having at least onealkoxide ion or thiolate ion, respectively) can be used in accordancewith the invention and the invention is not limited in this regard.Although water-soluble polymers bearing only a single hydroxyl or thiolcan be used, polymers bearing two, three, four, five, six, seven, eight,nine, ten, eleven, twelve or more hydroxyl and/or thiol moieties can beused. Advantageously, as the number of hydroxyl or thiol moieties on thewater-polymer segment increases, the number of available sites forproviding carboxylic acid moieties increases. Nonlimiting examples ofthe upper limit of the number of hydroxyl and/or thiol moietiesassociated with the water-soluble polymer segment include 500, 100, 80and 40.

The water-soluble polymer segment is preferably, although notnecessarily, a poly(ethylene glycol) or “PEG” or a derivative thereof.It should be understood, however, that related polymers are also suitedfor use in the practice of this invention and that the use of the term“PEG” or “poly(ethylene glycol)” is intended to be inclusive and notexclusive in this respect. Consequently, the term “PEG” includespoly(ethylene glycol) in any of its linear, branched or multi-arm forms,including alkoxy PEG, bifunctional PEG, forked PEG, branched PEG,pendant PEG, or PEG with degradable linkages therein, to be more fullydescribed below.

In one form useful in the present invention, free or non-bound PEG is alinear polymer terminated at each end with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—OH(m′) typically ranges from zero to about 4,000, preferably about 20 toabout 1,000.

The above polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the-PEG-symbol can represent the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—where (m′) is as defined as above.

Another type of PEG useful in the present invention is methoxy-PEG-OH,or mPEG in brief, in which one terminus is the relatively inert methoxygroup, while the other terminus is a hydroxyl group. The structure ofMPEG is given below.CH₃O—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—OHwhere (m′) is as described above.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, can also be used as the PEG polymer. For example,PEG can have the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones (either the same or different),such as methoxy poly(ethylene glycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine.

In addition, the PEG can comprise a forked PEG. An example of a forkedPEG is represented by the following structure:

wherein: X is a spacer moiety and each Z is an activated terminal grouplinked to CH by a chain of atoms of defined length. InternationalApplication No. PCT/US99/05333, discloses various forked PEG structurescapable of use in the present invention. The chain of atoms linking theZ functional groups to the branching carbon atom serve as a tetheringgroup and may comprise, for example, alkyl chains, ether chains, esterchains, amide chains and combinations thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG rather than at the end of the PEG chain. The pendant reactive groupscan be attached to the PEG directly or through a spacer moiety, such asan alkylene group.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more weak or degradable linkages in the polymer,including any of the above described polymers. For example, PEG can beprepared with ester linkages in the polymer that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O

-PEG-CO₂H+HO-PEG-.

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al. (1997) Polymer Preprints 38(1):582-3); phosphateester linkages formed, for example, by reacting an alcohol with aphosphate group; hydrazone linkages which are typically formed byreaction of a hydrazide and an aldehyde; acetal linkages that aretypically formed by reaction between an aldehyde and an alcohol; orthoester linkages that are, for example, formed by reaction between aformate and an alcohol; amide linkages formed by an amine group, e.g.,at an end of a polymer such as PEG, and a carboxyl group of another PEGchain; urethane linkages formed from reaction of, e.g., a PEG with aterminal isocyanate group and a PEG alcohol; peptide linkages formed byan amine group, e.g., at an end of a polymer such as PEG, and a carboxylgroup of a peptide; and oligonucleotide linkages formed by, for example,a phosphoramidite group, e.g., at the end of a polymer, and a 5′hydroxyl group of an oligonucleotide.

It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the above formsof PEG.

Many other polymers are also suitable for the invention. Polymers thatare non-peptidic and water-soluble, with from 2 to about 300 termini,are particularly useful in the invention. Examples of suitable polymersinclude, but are not limited to, other poly(alkylene glycols), such aspoly(propylene glycol) (“PPG”), copolymers of ethylene glycol andpropylene glycol and the like, poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), such as described in U.S. Pat. No.5,629,384, and copolymers, terpolymers, and mixtures thereof. Thesepolymers may be linear, or may be in any of the above-described forms(e.g., branched, forked, and the like).

Although the nominal average molecular weight of the water-solublepolymer can vary, the nominal average molecular weight will typically bein one or more of the following ranges: about 100 Daltons to about100,000 Daltons; from about 500 Daltons to about 80,000 Daltons; fromabout 1,000 Daltons to about 50,000 Daltons; from about 2,000 Daltons toabout 25,000 Daltons; from about 5,000 Daltons to about 20,000 Daltons.Exemplary nominal average molecular weights for the water-solublepolymer segment include about 1,000 Daltons, about 5,000 Daltons, about10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000Daltons, and about 30,000 Daltons.

The PEG and other water-soluble polymers as described herein aretypically considered to be biocompatible and non-immunogenic. Withrespect to biocompatibility, a substance is considered biocompatible ifthe beneficial effects associated with use of the substance alone orwith another substance (e.g., active agent) in connection with livingtissues (e.g., administration to a patient) outweighs any deleteriouseffects as evaluated by a clinician, e.g., a physician. With respect tonon-immunogenicity, a substance is considered non-immunogenic if use ofthe substance alone or with another substance in connection with livingtissues does not produce an immune response (e.g., the formation ofantibodies) or, if an immune response is produced, that such a responseis not deemed clinically significant or important as evaluated by aclinician. It is particularly preferred that the polymers describedherein as well as conjugates of active agents and the water-solublepolymers and segments described herein are biocompatible andnon-immunogenic.

Those of ordinary skill in the art will recognize that the foregoingdiscussion concerning substantially water-soluble polymers is by nomeans exhaustive and is merely illustrative, and that all polymericmaterials having the qualities described above are contemplated. As usedherein, the “term water-soluble polymer” generally refers to an entiremolecule, which can comprise functional groups such as hydroxyl groups,thiol groups, ortho ester functionalities and so forth. The termwater-soluble polymer segment is generally reserved for use indiscussing specific molecular structures wherein a polymer or portionthereof is but one part of the overall molecular structure.

An example of a preferred water-soluble polymer bearing a hydroxyl orthiol moiety comprises the following structure:POLY-(X)_(a)—YH  (Formula II)wherein:

POLY is a water-soluble polymer segment;

(a) is zero or one;

X, when present, is a spacer moiety; and

Y is O or S.

Recognizing certain instances wherein a water-soluble polymer segment(i.e., a “POLY”) is defined as containing a hydroxyl or thiol moiety(e.g., CH₃O—(CH₂CH₂O)_(m)—H or CH₃O—(CH₂CH₂O)_(m)—(CH₂CH₂S)—H,respectively), the “—YH” moiety of Formula II is understood to representthe hydroxyl or thiol moiety of “POLY” and not the irrationalinterpretation of, for example, “CH₃O—(CH₂CH₂O)_(m)—H—YH.”Alternatively, CH₃O—(CH₂CH₂O)_(m)—H, for example, is encompassed byFormula II when POLY is defined as “CH₃O—(CH₂CH₂O)_(m)—,” (a) is one, Xis “—CH₂CH₂-” and Y is “—O—”. Thus, given the possibility that there canbe more than a single way for any individual molecule to be encompassedby a given formula, due consideration must be given in order todetermine whether a molecule in question is or is not encompassed by agiven formula.

A particular preferred water-soluble segment bearing a single hydroxylgroup comprises the following structure:R⁵—O—(CH₂CH₂O)_(m)—Hwherein:

(m) is from 2 to 4000; and

R⁵ is an end-capping group such as H or an organic radical selected fromthe group consisting of alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl. It isespecially preferred that R⁵ is a lower alkyl such as methyl, althoughbenzyl and other end-capping groups known to those of skill in the artcan also be used.

For purposes of the present disclosure, however, a series of atoms isnot a spacer moiety when the series of atoms is immediately adjacent toa polymer and the series of atoms is but another monomer such that theproposed spacer moiety would represent a mere extension of the polymerchain. For example, given the partial structure “POLY-X—,” and POLY isdefined as “CH₃O(CH₂CH₂O)_(m)—” wherein (m) is 2 to 4000 and X isdefined as a spacer moiety, the spacer moiety cannot be defined as“—CH₂CH₂O—” since such a definition would merely represent an extensionof the polymer. In such a case, however, an acceptable spacer moietycould be defined as “—CH₂CH₂—.”

Exemplary spacer moieties include, but are not limited to, —C(O)—,—C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—,—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—,—CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,—CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]_(h)—(OCH₂CH₂)_(j)—, bivalent cycloalkyl group, —O—,—S—, an amino acid, —N(R⁶)—, and combinations of two or more of any ofthe foregoing, wherein R⁶ is H or an organic radical selected from thegroup consisting of alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) iszero to six, and (j) is zero to 20. Other specific spacer moieties havethe following structures: —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, and —O—C(O)—NH—(CH₂)₁ ₋₆—NH—C(O)—,wherein the subscript values following each methylene indicate thenumber of methylenes contained in the structure, e.g., (CH₂)₁₋₆ meansthat the structure can contain 1, 2, 3, 4, 5 or 6 methylenes.

In the present context of an amino acid being included in the structuresprovided herein, it should be remembered that the amino acid isconnected to the rest of the structure via one, two, three or moresites. For example, a spacer moiety can result when an amino acid isattached to the rest of the molecule via two covalent attachments. Inaddition, a branching structure can result when an amino acid isattached to the rest of the molecule via three sites. Thus, the aminoacid structure necessarily changes somewhat due to the presence of oneor more covalent attachments (e.g., removal of a hydrogen atom from theamino acid in order to accommodate a covalent linkage). Consequently,reference to an “amino acid” therefore includes the amino acidcontaining one or more linkages to other atoms. The amino acid can beselected from the group consisting of alanine, arginine, asparagines,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine. Both the D and L forms ofthe amino acids are contemplated.

The Ortho Ester of a Water-Soluble Polymer

The present invention provides for water-soluble polymers comprising anortho ester functionality. As described above, both acyclic and cyclicforms of the ortho ester functionality are included. Exemplary cyclicortho esters of water-soluble polymers of the invention are shown below:

wherein (m) is 2 to 4000, (a) is zero or one, and X¹, when present, is aspacer moiety. Of course, other water-soluble polymers comprising anortho ester moiety are also possible and in accordance with the presentinvention.

The Water-Soluble Polymer Bearing a Terminal Carboxylic Acid or EsterThereof

In accordance with the present methods, any number of polymers bearing aterminal carboxylic acid or ester thereof can be prepared and theinvention is not limited in this regard. Consequently, the inventionincludes carboxylic acids, such as alkanoic acids, and the correspondingesters of a polymer formed by a method as provided herein.

With respect to alkanoic acids then, the invention provides for alkanoicacids comprising the following structure:

wherein:

POLY is a water-soluble polymer segment;

(a) is either zero or one;

X, when present, is a spacer moiety;

(z) is an integer from 1 to 24;

R¹, in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl; and

R², in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl.

In addition, esters of the water-soluble polymer bearing a terminalcarboxylic acid are provided. For example, the corresponding esters ofthe carboxylic acids of Formula V preferably have the followingstructure:

wherein POLY, (a), X, when present, (z), each R¹, and each R² are aspreviously defined, and R³ is an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, and substituted aryl. In order torefer to polymers bearing a terminal carboxylic acid or ester thereof,R³ can conveniently be defined as H (thereby referring to the acid) oran organic radical selected from the group consisting of alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, and substituted aryl (thereby referring to acorresponding ester). The carboxylic acids and esters provided hereininclude sulfur-substituted versions, e.g., —C(O)—S—R³, as well.

For any given carboxylic acid, the corresponding ester can be formedusing conventional techniques. For example, the water-soluble polymerbearing a terminal carboxylic acid can undergo acid-catalyzedcondensation with an alcohol, thereby providing the corresponding ester.One approach to accomplish this is to use the method commonly referredto as a Fischer esterification reaction. Other techniques for forming adesired ester are known by those of ordinary skill in the art.

In addition, the water-soluble polymer bearing a terminal carboxylicacid can be modified to form useful reactive derivatives of alkanoicacids using methodology known in the art. For example, the carboxylicacid can be further derivatized to form acyl halides, acylpseudohalides, such as acyl cyanide, acyl isocyanate, and acyl azide,neutral salts, such as alkali metal or alkaline-earth metal salts (e.g.calcium, sodium, and barium salts), esters, anhydrides, amides, imides,hydrazides, and the like. In a preferred embodiment, the carboxylic acidis esterified to form an N-succinimidyl ester, o-, m-, or p-nitrophenylester, 1-benzotriazolyl ester, imidazolyl ester, or N-sulfosuccinimidylester. For example, the carboxylic acid can be converted into thecorresponding N-succinimidyl ester by reacting the carboxylic acid withdicyclohexyl carbodiimide (DCC) or diisopropyl carbodiimide (DIC) in thepresence of a base.

Particularly preferred polymers bearing a terminal carboxylic acidmoiety, however, are those wherein the carboxylic acid moiety forms partof an alkanoic acid. In this respect, carbon chains of four or morecarbon atoms (including the carbonyl carbon) that terminate in acarboxylic acid or nonaromatic ester are preferred. It is preferred thatthe water-soluble polymer segment is covalently attached through one ormore atoms to the distal carbon (with respect to the carbonyl carbon) inthe carbon chain that is at least four carbon atoms. Moreover, when thewater-soluble polymer segment is covalently attached through only oneatom, the one atom is not O or S. Such polymers can be structurallydefined as follows:

wherein:

POLY is a water-soluble polymer segment;

X¹ is a spacer moiety with the proviso that when the spacer moiety isonly one atom, the one atom is not O or S;

(z′) is an integer from 3 to 21;

R¹, in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl;

R², in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl; and

R⁷ is H or an organic radical selected from the group consisting ofalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, andsubstituted alkynyl.

Preferably, (z′) is three, four, or five. When (z′) is equal to three,the polymer of Formula VIa is comprised of the following structure:

wherein POLY, X′, each R¹, each R² and R⁷ are as previously defined.

When (z′) is equal to four, the polymer of Formula VIa is comprised ofthe following structure:

wherein POLY, X′, each R¹, each R² and R⁷ are as previously defined.

When (z′) is equal to five, the polymer of Formula VIa is comprised ofthe following structure:

wherein POLY, X′, each R¹, each R² and R⁷ are as previously defined.

In each of these cases, R⁷ is hydrogen when the carboxylic acid isdesired. With respect to R¹ and R², in some instances, each R¹ and R² ishydrogen. In other instances, however, the R¹ attached to the carbon ato the carbonyl carbon is alkyl (e.g., methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl) and all otherR¹ variables are H and all R² variables are hydrogen. As is known bythose of ordinary skill in the art, the “carbon a to the carbonyl”indicates the carbon atom directly attached to the carbonyl carbon. Forillustrative purposes, the “carbon a to the carbonyl carbon” as well asthe “carbonyl carbon” are labeled in the following structure:

Other arrangements are also envisioned wherein R¹ attached to the carbonatom β, γ or δ, to the carbonyl carbon is an organic radical. Inaddition, combinations in which two or more R¹ substituents are definedas an organic radical are possible. These same substitutions apply tothe corresponding ortho ester polymers and reagents.

When the carbon a to the carbonyl carbon bears an organic radical (e.g.,methyl), the resulting polymer may comprise a chiral center. Specificchirality, however, is not explicitly illustrated herein with respect toany compound or structure comprising one or more chiral centers and theinvention is intended to encompass both the isomerically pure forms ofthe compound or structure as well as diastereomeric mixtures, includinga racemic mixture, thereof. X′ is the same as X, as defined above, withthe exception that X′ is not O or S.

The carboxylic acids as provided herein may also be defined through thefollowing formula:

wherein:

R¹⁰ is H or an organic radical selected from the group consisting ofalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, and substituted aryl;

Q is a residue of a polyhydric alcohol having x+y hydroxyl groups;

R⁸, in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl;

R⁹, in each occurrence, is independently H or an organic radicalselected from the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substitutedaryl;

(x) is 1 to 20, preferably 2, 3, 4, 5, or 6;

(y) is 1 to 20; and

(z) is 1 to 24, preferably from 4 to 20.

Representative Q moieties include the following: propylene glycol,glycerine, sorbitol, pentaerythritol, dipentaerythritol,dihydroxycyclohexane, glucose, galactose, mannose, fructose, mannose,lactose, sucrose, amylose, as well as other sugars.

An example preferred polymer bearing two terminal carboxylic acidmoieties (a “forked” structure) comprises the following:

wherein (m) is from 2 to 4000. Preferably, however, the weight averagemolecular weight for the water-soluble polymer segment is from about5,000 Daltons to about 40,000 Daltons, more preferably from about 20,000Daltons to about 30,000 Daltons, with a molecular weight of about 20,000Daltons being most preferred.

Storage Conditions Generally

The polymers bearing a terminal carboxylic acid or ester thereof, aswell as any intermediates in their formation (e.g., ortho esters ofwater-soluble polymers), can be stored under an inert atmosphere, suchas under argon or under nitrogen. In this way, potentially degradativeprocesses associated with, for example, atmospheric oxygen, are avoidedor reduced entirely. In some cases, to avoid oxidative degradation,antioxidants, such as butylated hydroxyl toluene (BHT), can be added tothe final product prior to storage. In addition, it is preferred tominimize the amount moisture associated with the storage conditions toreduce potentially damaging reactions associated with water. Moreover,it is preferred to keep the storage conditions dark in order to preventcertain degradative processes that involve light. Thus, preferredstorage conditions include one or more of the following: storage underdry argon or another dry inert gas; storage at temperatures below about−15° C.; storage in the absence of light; and storage with a suitableamount (e.g., about 50-500 parts per million) of an antioxidant such asBHT.

Conjugation Method

The above-described polymers bearing a terminal carboxylic acid,optionally in an activated form, are useful for conjugation tobiologically active agents or surfaces comprising at least one groupsuitable for reaction with a carboxylic acid or the optional activatedform. Exemplary groups suitable for reaction with a carboxylic acidinclude amino groups (e.g., primary amines), hydrazines, hydrazides, andalcohols. Often, the polymer bearing a terminal carboxylic acid moietycan be conjugated directly to the active agent or surface. Sometimes,however, it is necessary to form an “activated” version of thecarboxylic acid in order to enhance reactivity to the biologicallyactive agent or surface. Methods for activating carboxylic acids areknown in the art and include, for example, dissolving the water-solublepolymer bearing a terminal carboxylic acid in methylene chloride andsubsequently adding N-hydroxysuccinimide andN,N-dicyclohexylcarbodiimide (DCC) to form an activated N-succinimidylester version of the carboxylic acid. Other approaches for activating acarboxylic acid are known to those of ordinary skill in the art.

Typically, the water-soluble polymer bearing the carboxylic acid orester thereof is added to the active agent or surface at an equimolaramount (with respect to the desired number of groups suitable forreaction with the carboxylic acid or ester thereof) or at a molarexcess. For example, the polymer can be added to the target active agentat a molar ratio of about 1:1 (polymer:active agent), 1.5:1, 2:1, 3:1,4:1, 5:1, 6:1, 8:1, or 10:1. The conjugation reaction is allowed toproceed until substantially no further conjugation occurs, which cangenerally be determined by monitoring the progress of the reaction overtime. Progress of the reaction can be monitored by withdrawing aliquotsfrom the reaction mixture at various time points and analyzing thereaction mixture by SDS-PAGE or MALDI-TOF mass spectrometry or any othersuitable analytical method. Once a plateau is reached with respect tothe amount of conjugate formed or the amount of unconjugated polymerremaining, the reaction is assumed to be complete. Typically, theconjugation reaction takes anywhere from minutes to several hours (e.g.,from 5 minutes to 24 hours or more). The resulting product mixture ispreferably, but not necessarily purified, to separate out excessreagents, unconjugated reactants (e.g., active agent) undesiredmulti-conjugated species, and free or unreacted polymer. The resultingconjugates can then be further characterized using analytical methodssuch as MALDI, capillary electrophoresis, gel electrophoresis, and/orchromatography.

Characterization/Separation

With respect to polymer-active agent conjugates, the conjugates can bepurified to obtain/isolate different conjugated species. Alternatively,and more preferably for lower molecular weight (e.g., less than about 20kiloDaltons, more preferably less than about 10 kiloDaltons) polymers,the product mixture can be purified to obtain the distribution ofwater-soluble polymer segments per active agent. For example, theproduct mixture can be purified to obtain an average of anywhere fromone to five PEGs per active agent (e.g., protein), typically an averageof about 3 PEGs per active agent (e.g., protein). The strategy forpurification of the final conjugate reaction mixture will depend upon anumber of factors, including, for example, the molecular weight of thepolymer employed, the particular active agent, the desired dosingregimen, and the residual activity and in vivo properties of theindividual conjugate(s).

If desired, conjugates having different molecular weights can beisolated using gel filtration chromatography. That is to say, gelfiltration chromatography is used to fractionate differently numberedpolymer-to-active agent ratios (e.g., 1-mer, 2-mer, 3-mer, and so forth,wherein “1-mer” indicates 1 polymer to active agent, “2-mer” indicatestwo polymers to active agent, and so on) on the basis of their differingmolecular weights (where the difference corresponds essentially to theaverage molecular weight of the water-soluble polymer segments). Forexample, in an exemplary reaction where a 100 kDa protein is randomlyconjugated to a PEG alkanoic acid having a molecular weight of about 20kDa, the resulting reaction mixture will likely contain unmodifiedprotein (MW 100 kDa), mono-pegylated protein (MW 120 kDa), di-pegylatedprotein (MW 140 kDa), and so forth. While this approach can be used toseparate PEG and other polymer conjugates having different molecularweights, this approach is generally ineffective for separatingpositional isomers having different polymer attachment sites within theprotein. For example, gel filtration chromatography can be used toseparate from each other mixtures of PEG 1-mers, 2-mers, 3-mers, and soforth, although each of the recovered PEG-mer compositions may containPEGs attached to different reactive amino groups (e.g., lysine residues)within the active agent.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) optical density (OD) at280 nm for protein content, (ii) bovine serum albumin (BSA) proteinanalysis, (iii) iodine testing for PEG content [Sims et al. (1980) Anal.Biochem, 107:60-63], and (iv) sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS PAGE), followed by staining with barium iodide.

Separation of positional isomers is carried out by reverse phasechromatography using a reverse phase-high performance liquidchromatography (RP-HPLC) C18 column (Amersham Biosciences or Vydac) orby ion exchange chromatography using an ion exchange column, e.g., aSepharose™ ion exchange column available from Amersham Biosciences.Either approach can be used to separate polymer-active agent isomershaving the same molecular weight (positional isomers).

Storage Conditions of Polymer-Active Agent Conjugates

Following conjugation, and optionally additional separation steps, theconjugate mixture may be concentrated, sterile filtered, and stored atlow a temperature, typically from about −20° C. to about −80° C.Alternatively, the conjugate may be lyophilized, either with or withoutresidual buffer and stored as a lyophilized powder. In some instances,it is preferable to exchange a buffer used for conjugation, such assodium acetate, for a volatile buffer such as ammonium carbonate orammonium acetate, that can be readily removed during lyophilization, sothat the lyophilized powder is absent residual buffer. Alternatively, abuffer exchange step may be used using a formulation buffer, so that thelyophilized conjugate is in a form suitable for reconstitution into aformulation buffer and ultimately for administration to a mammal.

Active Agents and Surfaces

The water-soluble polymers bearing a carboxylic acid or ester thereofpresented herein, can be attached, either covalently or non-covalently,to a number of entities including films, chemical separation andpurification surfaces, solid supports, metal surfaces such as gold,titanium, tantalum, niobium, aluminum, steel, and their oxides, siliconoxide, macromolecules (e.g., proteins, polypeptides, and so forth), andsmall molecules. Additionally, the polymers can also be used inbiochemical sensors, bioelectronic switches, and gates. The polymers canalso be employed as carriers for peptide synthesis, for the preparationof polymer-coated surfaces and polymer grafts, to prepare polymer-ligandconjugates for affinity partitioning, to prepare cross-linked ornon-cross-linked hydrogels, and to prepare polymer-cofactor adducts forbioreactors.

A biologically active agent for use in coupling to a polymer aspresented herein may be any one or more of the following. Suitableagents can be selected from, for example, hypnotics and sedatives,psychic energizers, tranquilizers, respiratory drugs, anticonvulsants,muscle relaxants, antiparkinson agents (dopamine antagnonists),analgesics, anti-inflammatories, antianxiety drugs (anxiolytics),appetite suppressants, antimigraine agents, muscle contractants,anti-infectives (antibiotics, antivirals, antifungals, vaccines)antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxicants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

More particularly, the active agent may fall into one of a number ofstructural classes, including but not limited to small molecules(preferably insoluble small molecules), peptides, polypeptides,proteins, polysaccharides, steroids, nucleotides, oligonucleotides,polynucleotides, fats, electrolytes, and the like. Preferably, an activeagent for coupling to a polymer as described herein possesses a nativeamino group, or alternatively, is modified to contain at least onereactive amino group suitable for conjugating to a polymer describedherein.

Specific examples of active agents suitable for covalent attachmentinclude but are not limited to aspariginase, aindoxovir (DAPD), antide,becaplermin, calcitonins, cyanovirin, denileukin diftitox,erythropoietin (EPO), EPO agonists (e.g., peptides from about 10-40amino acids in length and comprising a particular core sequence asdescribed in WO 96/40749), dornase alpha, erythropoiesis stimulatingprotein (NESP), coagulation factors such as Factor V, Factor VII, FactorVIIa, Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII, vonWillebrand factor; ceredase, cerezyme, alpha-glucosidase, collagen,cyclosporin, alpha defensins, beta defensins, exedin-4, granulocytecolony stimulating factor (GCSF), thrombopoietin (TPO), alpha-1proteinase inhibitor, elcatonin, granulocyte macrophage colonystimulating factor (GMCSF), fibrinogen, filgrastim, growth hormoneshuman growth hormone (hGH), growth hormone releasing hormone (GHRH),GRO-beta, GRO-beta antibody, bone morphogenic proteins such as bonemorphogenic protein-2, bone morphogenic protein-6, OP-1; acidicfibroblast growth factor, basic fibroblast growth factor, CD-40 ligand,heparin, human serum albumin, low molecular weight heparin (LMWH),interferons such as interferon alpha, interferon beta, interferon gamma,interferon omega, interferon tau, consensus interferon; interleukins andinterleukin receptors such as interleukin-1 receptor, interleukin-2,interleukin-2 fusion proteins, interleukin-1 receptor antagonist,interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6,interleukin-8, interleukin-12, interleukin-13 receptor, interleukin-17receptor; lactoferrin and lactoferrin fragments, luteinizing hormonereleasing hormone (LHRH), insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin,C-peptide, somatostatin, somatostatin analogs including octreotide,vasopressin, follicle stimulating hormone (FSH), influenza vaccine,insulin-like growth factor (IGF), insulintropin, macrophage colonystimulating factor (M-CSF), plasminogen activators such as alteplase,urokinase, reteplase, streptokinase, pamiteplase, lanoteplase, andteneteplase; nerve growth factor (NGF), osteoprotegerin,platelet-derived growth factor, tissue growth factors, transforminggrowth factor-1, vascular endothelial growth factor, leukemia inhibitingfactor, keratinocyte growth factor (KGF), glial growth factor (GGF), TCell receptors, CD molecules/antigens, tumor necrosis factor (TNF),monocyte chemoattractant protein-1, endothelial growth factors,parathyroid hormone (PTH), glucagon-like peptide, somatotropin, thymosinalpha 1, rasburicase, thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta10, thymosin beta 9, thymosin beta 4, alpha-1 antitrypsin,phosphodiesterase (PDE) compounds, VLA-4 (very late antigen-4), VLA-4inhibitors, bisphosphonates, respiratory syncytial virus antibody,cystic fibrosis transmembrane regulator (CFTR) gene, deoxyreibonuclease(Dnase), bactericidal/permeability increasing protein (BPI), andanti-CMV antibody. Exemplary monoclonal antibodies include etanercept (adimeric fusion protein consisting of the extracellular ligand-bindingportion of the human 75 kD TNF receptor linked to the Fc portion ofIgG1), abciximab, afeliomomab, basiliximab, daclizumab, infliximab,ibritumomab tiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomabconjugate, olizumab, rituximab, trastuzumab (herceptin), and adalimumab.

Additional agents suitable for covalent attachment include, but are notlimited to, adefovir, alosetron, amifostine, amiodarone, aminocaproicacid, aminohippurate sodium, aminoglutethimide, aminolevulinic acid,aminosalicylic acid, amsacrine, anagrelide, anastrozole, aripiprazole,asparaginase, anthracyclines, bexarotene, bicalutamide, bleomycin,buserelin, busulfan, cabergoline, capecitabine, carboplatin, carmustine,chlorambucin, cilastatin sodium, cisplatin, cladribine, clodronate,cyclophosphamide, cyproterone, cytarabine, camptothecins, 13-cisretinoic acid, all trans retinoic acid; dacarbazine, dactinomycin,daunorubicin, deferoxamine, dexamethasone, diclofenac,diethylstilbestrol, docetaxel, doxorubicin, dutasteride, epirubicin,estramustine, etoposide, exemestane, ezetimibe, fexofenadine,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,fondaparinux, fulvestrant, gamma-hydroxybutyrate, gemcitabine,epinephrine, L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib,irinotecan, itraconazole, goserelin, letrozole, leucovorin, levamisole,lisinopril, lovothyroxine sodium, lomustine, mechlorethamine,medroxyprogesterone, megestrol, melphalan, mercaptopurine, metaraminolbitartrate, methotrexate, metoclopramide, mexiletine, mitomycin,mitotane, mitoxantrone, naloxone, nicotine, nilutamide, nitisinone,octreotide, oxaliplatin, pamidronate, pentostatin, pilcamycin, porfimer,prednisone, procarbazine, prochlorperazine, ondansetron, oxaliplatin,raltitrexed, sirolimus, streptozocin, tacrolimus, pimecrolimus,tamoxifen, tegaserod, temozolomide, teniposide, testosterone,tetrahydrocannabinol, thalidomide, thioguanine, thiotepa, topotecan,treprostinil, tretinoin, valdecoxib, celecoxib, rofecoxib, valrubicin,vinblastine, vincristine, vindesine, vinorelbine, voriconazole,dolasetron, granisetron; formoterol, fluticasone, leuprolide, midazolam,alprazolam, amphotericin B, podophylotoxins, nucleoside antivirals,aroyl hydrazones, sumatriptan; macrolides such as erythromycin,oleandomycin, troleandomycin, roxithromycin, clarithromycin, davercin,azithromycin, flurithromycin, dirithromycin, josamycin, spiromycin,midecamycin, loratadine, desloratadine, leucomycin, miocamycin,rokitamycin, andazithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin,netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, andstreptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,daptomycin, gramicidin, colistimethate; polymixins such as polymixin B,capreomycin, bacitracin, penems; penicillins includingpenicllinase-sensitive agents like penicillin G, penicillin V;penicllinase-resistant agents like methicillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, nafcillin; gram negative microorganismactive agents like ampicillin, amoxicillin, and hetacillin, cillin, andgalampicillin; antipseudomonal penicillins like carbenicillin,ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporinslike cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone,cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin,cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil,cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine,cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan,cefinetazole, ceftazidime, loracarbef, and moxalactam, monobactams likeaztreonam; and carbapenems such as imipenem, meropenem, and ertapenem,pentamidine isetionate, albuterol sulfate, lidocaine, metaproterenolsulfate, beclomethasone diprepionate, triamcinolone acetamide,budesonide acetonide, fluticasone, ipratropium bromide, flunisolide,cromolyn sodium, and ergotamine tartrate; taxanes such as paclitaxel;SN-38, and tyrphostines.

Preferred small molecules for coupling to a polymer as described hereinare those having at least one naturally occurring amino group. Preferredmolecules such as these include aminohippurate sodium, amphotericin B,doxorubicin, aminocaproic acid, aminolevulinic acid, aminosalicylicacid, metaraminol bitartrate, pamidronate disodium, daunorubicin,levothyroxine sodium, lisinopril, cilastatin sodium, mexiletine,cephalexin, deferoxamine, and amifostine.

Preferred peptides or proteins for coupling to a polymer as describedherein include EPO, IFN-α, IFN-β, consensus IFN, Factor VIII, Factor IX,GCSF, GMCSF, hGH, insulin, FSH, and PTH.

The above exemplary biologically active agents are meant to encompass,where applicable, analogues, agonists, antagonists, inhibitors, isomers,and pharmaceutically acceptable salt forms thereof. In reference topeptides and proteins, the invention is intended to encompass synthetic,recombinant, native, glycosylated, and non-glycosylated forms, as wellas biologically active fragments thereof.

Pharmaceutical Compositions

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium famerate, and combinationsthereof.

The pharmaceutical preparations encompass all types of formulations andin particular those that are suited for injection, e.g., powders thatcan be reconstituted as well as suspensions and solutions. The amount ofthe conjugate (i.e., the conjugate formed between the active agent andthe polymer described herein) in the composition will vary depending ona number of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container (e.g., avial). In addition, the pharmaceutical preparation can be housed in asyringe. A therapeutically effective dose can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about5%-98% by weight, more preferably from about 15-95% by weight of theexcipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical preparations of the present invention are typically,although not necessarily, administered via injection and are thereforegenerally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

As previously described, the conjugates can be administered injectedparenterally by intravenous injection, or less preferably byintramuscular or by subcutaneous injection. Suitable formulation typesfor parenteral administration include ready-for-injection solutions, drypowders for combination with a solvent prior to use, suspensions readyfor injection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

Methods of Administering

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with conjugate. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalpreparation). The method of administering may be used to treat anycondition that can be remedied or prevented by administration of theparticular conjugate. Those of ordinary skill in the art appreciatewhich conditions a specific conjugate can effectively treat. The actualdose to be administered will vary depend upon the age, weight, andgeneral condition of the subject as well as the severity of thecondition being treated, the judgment of the health care professional,and conjugate being administered. Therapeutically effective amounts areknown to those skilled in the art and/or are described in the pertinentreference texts and literature. Generally, a therapeutically effectiveamount will range from about 0.001 mg to 100 mg, preferably in dosesfrom 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10mg/day to 50 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

One advantage of administering the conjugates of the present inventionis that individual water-soluble polymer portions can be cleaved off.Such a result is advantageous when clearance from the body ispotentially a problem because of the polymer size. Optimally, cleavageof each water-soluble polymer portion is facilitated through the use ofphysiologically cleavable and/or enzymatically degradable linkages suchas urethane, amide, carbonate or ester-containing linkages. In this way,clearance of the conjugate (via cleavage of individual water-solublepolymer portions) can be modulated by selecting the polymer molecularsize and the type functional group that would provide the desiredclearance properties. One of ordinary skill in the art can determine theproper molecular size of the polymer as well as the cleavable functionalgroup. For example, one of ordinary skill in the art, using routineexperimentation, can determine a proper molecular size and cleavablefunctional group by first preparing a variety of polymer derivativeswith different polymer weights and cleavable functional groups, and thenobtaining the clearance profile (e.g., through periodic blood or urinesampling) by administering the polymer derivative to a patient andtaking periodic blood and/or urine sampling. Once a series of clearanceprofiles have been obtained for each tested conjugate, a suitableconjugate can be identified.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the experimental that follow areintended to illustrate and not limit the scope of the invention. Otheraspects, advantages and modifications within the scope of the inventionwill be apparent to those skilled in the art to which the inventionpertains.

All articles, books, patents, patent publications and other publicationsreferenced herein are hereby incorporated by reference in theirentireties.

EXPERIMENTAL Examples

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis and the like, which areunderstood by one of ordinary skill in the art and are explained in theliterature. In the following examples, efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperatures, andso forth), but some experimental error and deviation should be accountedfor. Unless otherwise indicated, temperature is in degrees Celsius andpressure is at or near atmospheric pressure at sea level. All reagentswere obtained commercially unless otherwise indicated. All generated NMRwas obtained from a 300 or 400 MHz NMR spectrometer manufactured byBruker (Billerica, Mass.). Reference to an “OBO ortho ester” correspondsto esters comprising the 4-methyl-2,6,7-trioxabicyclo[2.2.2]octanyl.

Example 1 Formation of 4-Bromobutyrate ester of3-methyl-3-hydroxymethyloxetane (MW=251.12)

3-Methyl-3-hydroxymethyloxetane (10.2 g, 0.1 mole) (Sigma-AldrichCorporation, St. Louis, Mo.) was dissolved in anhydrous dichloromethane(200 ml). Pyridine (9.8 ml, 0.12 moles) was then added to the solution.Thereafter, the solution was cooled to 0° C. and 4-bromobutyryl chloride(18.5 g, 0.1 mole) (Sigma-Aldrich Corporation, St. Louis, Mo.) dissolvedin anhydrous dichloromethane (50 ml) was added dropwise over 20 minutes.The mixture was stirred overnight under argon atmosphere. Next, thereaction mixture was washed with water and dried with anhydrousmagnesium sulfate. The solvent was then distilled off under reducedpressure. Yield 23.6 g. NMR (d₆-DMSO): 1.26 ppm (s, 3H), 2.07 ppm (m,2H), 2.51 ppm (t, 2H), 3.56 ppm (t, 2H), 4.14 ppm (s, 2H), 4.24 ppm (d,2H), 4.38 ppm (d, 2H).

Example 2 Formation of1-(3-Bromopropyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (MW=251.12)

The product of Example 1 (crude 4-bromobutyrate ester of3-methyl-3-hydroxymethyloxetane, 20.1 g, 0.08 moles) was dissolved inanhydrous dichloromethane (100 ml), the solution was cooled to 0° C. andboron trifluoride diethyl etherate (2.5 ml, 0.022 moles) was added. Themixture was then stirred for four hours at 0° C. Triethylamine (12 ml)was added, the mixture was stirred for 15 minutes, and the solvent wasdistilled off under reduced pressure. The crude product was dissolved inethyl ether (180 ml) and the solution was then filtered to remove thesolid impurities. Next, ether was distilled off and the product wasdistilled under reduced pressure (kugelrohr, 110-115° C., 0.05 mm Hg).Yield 15.0 g. NMR (d₆-DMSO): 0.74 ppm (s, 3H), 1.68 ppm (m, 2H), 1.88ppm (m, 2H), 3.52 ppm (t, 2H), 3.81 ppm (s, 6H).

Example 3 Synthesis of a PEG-Butanoic Acid Precursor Useful in aPolymerization Reaction

A mixture of anhydrous ethylene glycol (120 g, 1.93 moles), 1.0Msolution of potassium tert-butoxide in tert-butanol (70 ml, 0.070moles), and the product of Example 2[1-(3-bromopropyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane (15 g,0.060 moles)] was stirred overnight at 70° C. under an argon atmosphere.After cooling to room temperature, the reaction mixture was added to 600ml of distilled water. The product was three-times extracted withdichloromethane (150 ml, 125 ml, and 125 ml). The combined extracts weredried with anhydrous magnesium sulfate and the solvent was distilled offunder reduced pressure. The product (Compound 1) was then subjected tovacuum distillation (kugelrohr, t=120-130° C., 0.05 mm Hg). Yield 6.2 g.NMR (d₆-DMSO): 0.74 ppm (s, 3H), 1.59 ppm (m, 4H), 3.34 ppm (m, 4H),3.45 ppm (t, 2H), 3.80 ppm (s, 6H), 4.54 ppm (t, 1H).

Schematically, the reaction can be represented as follows:

Example 4 Synthesis of a PEG-Propanoic Acid Precursor Useful in aPolymerization Reaction

Tert-butyl acrylate (130 g, 1.01 mole) was added dropwise over 3 hoursto a mixture of anhydrous ethylene glycol (62 g, 1.0 mole),tetrabutylammonium bromide (9.6 g) and KOH (powder, 2.2 g), and stirredovernight at room temperature under an argon atmosphere. The volatileproducts were distilled off under reduced pressure (rotoevaporator, 60°C.) and the mixture was dissolved in 250 ml dichloromethane. Thesolution was washed with 250 ml of distilled water, dried with anhydrousmagnesium sulfate, and the solvent was distilled off under reducedpressure. The product (Compound 2) was then subjected to vacuumdistillation (kugelrohr, t=95-100° C., 0.05 mm Hg). Yield 36.6 g. NMR(d₆-DMSO): 1.40 ppm (s, 9H), 2.42 ppm (t, 2H), 3.39 ppm (m, 2H), 3.46ppm (m, 2H), 3.59 ppm (s, 2H), 4.55 ppm (t, 1H).

Schematically, the reaction can be represented as follows:

A mixture of Compound 2 (36.6 g, 0.19 moles), pyridine (52 ml, 0.64moles), acetic anhydride (52 ml, 0.55 moles) and dimethylaminopyridine(DMAP, 1.0 g) was stirred overnight at room temperature. The volatileproducts were then distilled off under reduced pressure (rotoevaporator,t=65° C.) and the product (Compound 3) was subjected to vacuumdistillation (kugelrohr, 100-110° C., 0.05 mm Hg). Yield 40.9 g. NMR(d₆-DMSO): 1.40 ppm (s, 9H), 2.02 ppm (s, 3H), 2.42 ppm (t, 2H), 3.58ppm (bm, 4H), 4.08 ppm (m, 2H).

Schematically, the reaction can be represented as follows:

To compound 3 (30.0 g, 0.19 moles), trifluoroacetic acid (40 ml) wasadded and the solution was stirred for 1 hour at room temperature. Thevolatile products were then distilled off under reduced pressure(rotoevaporator, t=60° C.) and the product was dissolved in 400 mldichloromethane. The solution was washed twice with 5% NaCl solution anddried with anhydrous magnesium sulfate and the solvent was distilled offunder reduced pressure to provide Compound 4. Yield 19.1 g. NMR(d₆-DMSO): 2.01 ppm (s, 3H), 2.44 ppm (t, 2H), 3.57 ppm (m, 2H), 3.61ppm (t, 2H) 4.09 ppm (m, 2H).

Schematically, the reaction can be represented as follows:

To a solution of compound 4 (19.1 g, 0.108 moles),3-methyl-3-hydroxyoxetane (17.6 g, 0.172 moles), 1-hydroxybenzotriazole(HOBt, 1.6 g), and DMAP (3.6 g) in anhydrous dichloromethane (500 ml),along with 1,3-dicyclohexylcarbodiimide (DCC, 1.0M solution indichloromethane, 114 ml, 0.114 moles) was added at 0° C., and themixture was stirred overnight at room temperature. The mixture was thenfiltered to remove precipitated 1,3-dicyclohexylurea and the solutionwas washed with 250 ml of 5% H₃PO₄. Next, the dichloromethane wasdistilled off under reduced pressure (rotoevaporator) and the product(Compound 5) was subjected to vacuum distillation (kugelrohr, 125-135°C., 0.05 mm Hg). Yield 18.5 g. NMR (d₆-DMSO): 1.26 ppm (s, 3H), 2.00 ppm(s, 3H), 2.59 ppm (t, 2H), 3.57 ppm (m, 2H), 3.66 ppm (t, 2H), 4.08 ppm(m, 2H), 4.14 ppm (s, 2H), 4.23 ppm (d, 2H), 4.38 ppm (d, 2H).

Schematically, the reaction can be represented as follows:

Compound 5 (15.0 g, 0.08 moles) was then dissolved in anhydrousdichloromethane (75 ml), the solution was cooled to 0° C. and borontrifluoride diethyl etherate (1.65 ml) was added. The mixture was thenstirred for 3 hours at 0° C. Triethylamine (7.5 ml) was added, themixture was stirred for 10 minutes, and the solvent was distilled offunder reduced pressure. The crude product (Compound 6) was dissolved inethyl ether (150 ml) and the solution was filtered to remove the solidimpurities. The ether was then distilled off. Yield 12.9 g. NMR(d₆-DMSO): 0.74 ppm (s, 3H), 1.83 ppm (t, 2H), 2.00 ppm (s, 3H), 3.46ppm (t, 2H), 3.52 ppm (m, 2H), 3.80 ppm (s, 2H), 3.52 ppm (t, 6H), 4.07ppm (m, 2H).

Schematically, the reaction can be represented as follows:

A mixture of compound 6 (12 g), ethyl alcohol (80 ml), and 50% aqueoussolution of potassium hydroxide (8 g) was stirred for 40 minutes at roomtemperature. The solvent was then distilled off under reduced pressure(rotoevaporator). The crude product was dissolved in 400 mldichloromethane and the solution was washed with 5% aqueous solution ofsodium chloride. Next, the solution was dried with anhydrous MgSO₄ andthe solvent was distilled off under reduced pressure (rotoevaporator)giving 8.0 g of colorless liquid product (Compound 7). NMR (d₆-DMSO):0.74 ppm (s, 3H), 1.83 ppm (t, 2H), 3.35 ppm (m, 2H), 3.46 ppm (m, 4H),3.80 ppm (s, 6H), 3.52 ppm (t, 6H), 4.54 ppm (t, 1H).

Schematically, the reaction can be represented as follows:

Example 5 Formation of PEG_((3,500 Da))-α-hydroxy-ω-butanoic Acid, OBOOrtho Ester Using Compound 1 as the Initiator for Polymerization

Compound 1 (0.564 g, 0.00243 moles), tetrahydrofuran (THF, 200 ml), andpotassium naphthalene, 0.3 mol/1-tetrahydrofuran solution (10 ml,0.00300 moles) were added to a glass reactor and stirred for 3 minutesin an argon atmosphere. Ethylene oxide (8.8 g, 0.20 moles) was added tothis solution and the reaction mixture was stirred for 44 hours at roomtemperature. Next, the mixture was purged with argon and 0.1M phosphatebuffer (pH=8, 100 ml) was added. The THF layer was separated anddiscarded. Naphthalene was removed from the solution by ethyl etherextraction. The product was then extracted with dichloromethane (3×50ml). The extract was dried with anhydrous sodium sulfate and the solventwas distilled off under reduced pressure. Yield 7.2 g. NMR (d₆-DMSO):0.73 ppm (s, —CH₃ of OBO, 3H), 1.57 ppm (m, —CH₂—CH₂—CO—, 4H), 3.51 ppm(s, PEG backbone), 3.80 ppm (s, CH₂ of OBO, 6H), 4.58 ppm (t, —OH, 1H).

Example 6 Formation of PEG_((3,500 Da))-α-hydroxy-ω-butanoic Acid

The product of Example 5 (i.e., PEG_((3,500 Da))-α-hydroxy-ω-butanoicacid, OBO ortho ester, 7.0 g) was dissolved in distilled water (100 ml).The pH of the solution was adjusted to 2 with 5% phosphoric acid and thesolution was stirred for 15 minutes at room temperature. Next, the pHwas readjusted to 12 by adding 1M sodium hydroxide and the solution wasstirred for two hours while maintaining a pH equal to 12 by periodicaddition of 1M sodium hydroxide. The pH was then adjusted to 3 with 5%phosphoric acid, after which the product was extracted withdichloromethane. The extract was dried with anhydrous magnesium sulfateand added to ethyl ether. The precipitated product was filtered off anddried under reduced pressure. Yield 6.6 g. NMR (d₆-DMSO): 1.72 ppm (q,CH₂—CH₂—COO—, 2H) 2.24 ppm (t, —CH₂—COO—, 2H), 3.51 ppm (s, PEGbackbone), 4.58 ppm (t, —OH, 1H).

Example 7 Formation of mPEG_((3,500 Da))-butanoic Acid OBO Ortho Ester

A mixture of the product of Example 5 (i.e.,PEG_((3,500 Da))-α-hydroxy-(D-butanoic acid, OBO ortho ester, 7.0 g,0.002 moles), toluene (100 ml), 1.0M solution of potassium tert-butoxidein tert-butanol (10 ml, 0.01 moles), and methyl p-toluenesulfonate (1.49g, 0.008 moles) was stirred overnight at 45° C. The solvents weredistilled off under reduced pressure (rotoevaporator). The crude productwas dissolved in dichloromethane and added to cold ethyl ether. Theprecipitated product was filtered off and dried under reduced pressure.Yield 6.2 g. NMR (d₆-DMSO): 0.73 ppm (s, —CH₃ of OBO, 3H), 1.57 ppm (m,—CH₂—CH₂—CO—, 4H), 3.24 ppm (s, —OCH₃, 3H), 3.51 ppm (s, PEG backbone),3.80 ppm (s, CH₂ of OBO, 6H).

Example 8 Formation of mPEG_((3,500 Da))-butanoic Acid

The product of Example 7 (mPEG_((3,500 Da))-butanoic acid, OBO orthoester, 6.0 g) was dissolved in distilled water (60 ml). The pH of thesolution was adjusted to 2 with 5% phosphoric acid and the solution wasstirred for 15 minutes at room temperature. Next, the pH was readjustedto 12 with 1M sodium hydroxide and the solution was stirred for 2 hours.The pH of 12 was maintained by periodic addition of 1M sodium hydroxide.After two hours of stirring and maintaining a pH of 12, the pH wasadjusted to 3 with 5% phosphoric acid and the product was extracted withdichloromethane. The extract was then dried with anhydrous magnesiumsulfate and added to ethyl ether. The precipitated product was filteredoff and dried under reduced pressure. Yield 5.6 g. NMR (d₆-DMSO): 1.72ppm (q, CH₂—CH₂—COO—, 2H) 2.24 ppm (t, —CH₂—COO—, 2H), 3.24 ppm (s,CH₃O—, 3H), 3.51 ppm (s, PEG backbone).

Example 9 Formation of PEG_((5,000 Da))-α-hydroxy-ω-propanoic Acid, OBOOrtho Ester Using Compound 7 as the Initiator for Polymerization

Compound 7 (0.53 g, 0.00243 moles), tetrahydrofuran (THF, 200 ml), andpotassium naphthalene 0.3 mol/1-tetrahydrofuran solution (10 ml, 0.00300moles) were added to a glass reactor and stirred for three minutes in anargon atmosphere. Ethylene oxide (12.2 g, 0.277 moles) was added to thissolution and the reaction mixture was stirred for 44 hours at roomtemperature. Next, the mixture was purged with argon and 0.1M phosphatebuffer (pH=8, 100 ml) was added. The THF layer was separated and thendiscarded. Naphthalene was removed from the solution by ethyl etherextraction. Thereafter, the product was extracted with dichloromethane(3×50 ml). The extract was dried with anhydrous sodium sulfate and thesolvent was distilled off under reduced pressure. Yield 11.7 g. NMR(d₆-DMSO): 0.73 ppm (s, —CH₃, 3H), 1.82 ppm (t, —CH₂—CO—, 2H), 3.51 ppm(s, PEG backbone), 3.80 ppm (s, CH₂ of OBO, 6H), 4.57 ppm (t, —OH, 1H).

Example 10 Formation of PEG_((5,000 Da))-α-hydroxy-ω-propanoic Acid

The product of Example 9 (PEG_((5,000 Da))-α-hydroxy-ω-propanoic acid,OBO ortho ester, 5.0 g) was dissolved in distilled water (75 ml). The pHof the solution was adjusted to 2 with 5% phosphoric acid and thesolution was stirred 15 minutes at room temperature. Next, the pH wasreadjusted to 12 with 1M sodium hydroxide and the solution was stirredfor two hours. The pH of the solution was maintained at a pH equaling 12by periodic addition of 1M sodium hydroxide. After two hours of stirringand maintaining the pH at 12, the pH of the solution was adjusted to 3with 5% phosphoric acid and the product was thereafter extracted withdichloromethane. The extract was then dried with anhydrous magnesiumsulfate and added to ethyl ether. The precipitated product was filteredoff and dried under reduced pressure. Yield 4.4 g. NMR (d₆-DMSO): 2.43ppm (t, —CH₂—COO—, 2H), 3.51 ppm (s, PEG backbone), 4.58 ppm (t, —OH,1H).

Example 11 Formation of mPEG_((5,000 Da))-propanoic Acid, OBO OrthoEster

A mixture of the product of Example 9(PEG_((5,000 Da))-α-hydroxy-ω-propanoic acid, OBO ortho ester, 4.0 g,0.0008 moles), toluene (50 ml), 1.0M solution of potassium tert-butoxidein tert-butanol (8 ml, 0.008 moles), and methyl p-toluenesulfonate (1.49g, 0.008 moles) was stirred overnight at 50° C. Next, the solvents weredistilled off under reduced pressure (rotoevaporator). The crude productwas then dissolved in dichloromethane and added to cold ethyl ether. Theprecipitated product was filtered off and dried under reduced pressure.Yield 3.6 g. NMR (d₆-DMSO): 0.73 ppm (s, —CH₃, 3H), 1.82 ppm (t,—CH₂—CO—, 2H), 3.24 ppm (s, CH₃O—, 3H), 3.51 ppm (s, PEG backbone), 3.80ppm (s, CH₂ of OBO, 6H).

Example 12 Formation of mPEG_((5,000 Da))-propanoic Acid

The product of Example 11 (mPEG_((5,000 Da))-propanoic acid, OBO orthoester, 6.0 g) was dissolved in distilled water (60 ml). The pH of thesolution was the adjusted to 2 with 5% phosphoric acid and the solutionwas stirred for 15 minutes at room temperature. Next, the pH wasreadjusted to 12 with 1M sodium hydroxide and the solution was stirredfor two hours. A pH of 12 was maintained by periodic addition of 1Msodium hydroxide. After two hours of stirring and maintaining a pH of12, the pH of the solution was then adjusted to 3 with 5% phosphoricacid and the product was then extracted with dichloromethane. Theextract was then dried with anhydrous magnesium sulfate and added toethyl ether. The precipitated product was filtered off and dried underreduced pressure. Yield 5.6 g. NMR (d₆-DMSO): 2.43 ppm (t, —CH₂—COO—,2H), 3.24 ppm (s, CH₃O—, 3H), 3.51 ppm (s, PEG backbone).

Example 13 Formation of mPEG_((20,000 Da))-butanoic Acid

A solution of mPEG_((20,000 Da)) (2.0 g, 0.0001 moles) (NOF Corporation)in toluene (30 ml) was azeotropically dried by distilling off 15 ml oftoluene. 1.0M solution of potassium tert-butoxide in tert-butanol (0.80ml, 0.0008000 moles) and the product of Example 2[1-(3-bromopropyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane, 0.15 g,0.0005973 moles] were added and the mixture was stirred overnight at 70°C. under argon atmosphere. The solvent was distilled off under reducedpressure and the residue was dissolved in distilled water (40 ml). ThepH of the solution was adjusted to 2 with 5% phosphoric acid and thesolution was stirred for 15 minutes at room temperature. Next, the pHwas readjusted to 12 with 1M sodium hydroxide and the solution wasstirred for two hours keeping the pH at 12 by periodic addition of 1Msodium hydroxide. Thereafter, the pH was adjusted to 3 with 5%phosphoric acid and the product was extracted with dichloromethane. Theextract was dried with anhydrous magnesium sulfate and added to ethylether. The precipitated product was filtered off and dried under reducedpressure. Yield 1.6 g. NMR (d₆-DMSO): 1.72 ppm (q, CH₂ —CH₂—COO—) 2.24ppm (t, —CH₂—COO—), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone).Anion exchange chromatography: mPEG_((20,000))-butanoic acid 98.6%,m-PEG-20K 1.4%.

Example 14 Formation of 4-Bromohexanoate Ester of3-Methyl-3-hydroxymethyloxetane (MW=251.12)

3-Methyl-3-hydroxymethyloxetane (20.5 g, 0.201 mole) was dissolved inanhydrous dichloromethane (250 ml) and pyridine (20.0 ml, 0.12 moles)was added. The solution was cooled to 0° C. and 4-bromohexanoyl chloride(42.7 g, 0.200 mole) dissolved in anhydrous dichloromethane (50 ml) wasadded dropwise over 20 minutes. Thereafter, the mixture was stirredovernight under argon atmosphere. Next, the reaction mixture was washedwith water and dried with anhydrous magnesium sulfate. The solvent wasthen distilled off under reduced pressure. Yield 56.8 g. NMR (d₆-DMSO):1.26 ppm (s, 3H), 2.07 ppm (m, 2H), 2.51 ppm (t, 2H), 3.56 ppm (t, 2H),4.14 ppm (s, 2H), 4.24 ppm (d, 2H), 4.38 ppm (d, 2H).

Example 15 Formation of1-(3-Bromopentyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane

The product of Example 14 (crude 4-bromohexanoate ester of3-methyl-3-hydroxymethyloxetane, 20.1 g, 0.08 moles) was dissolved inanhydrous dichloromethane (100 ml), the solution was cooled to 0° C.,and boron trifluoride diethyl etherate (2.5 ml, 0.022 moles) was added.The mixture was then stirred for 4 hours at 0° C. Triethylamine (12 ml)was added, the mixture was stirred for 15 minutes, and then the solventwas distilled off under reduced pressure. The crude product was thendissolved in ethyl ether (180 ml) and the solution was filtered toremove the solid impurities. Next, ether was distilled off and theproduct was distilled under reduced pressure (kugelrohr, 110-115° C.,0.05 mm Hg). Yield 15.0 g. NMR (d₆-DMSO): 0.74 ppm (s, 3H), 1.68 ppm (m,2H), 1.88 ppm (m, 2H), 3.52 ppm (t, 2H), 3.81 ppm (s, 6H).

Example 16 Formation of mPEG_((2,000 Da))-hexanoic Acid

A solution of mPEG_((2,000 Da)) (2.0 g, 0.0010 moles) (NOF Corporation)in toluene (30 ml) was azeotropically dried by distilling off 15 mltoluene. 1.0M solution of potassium tert-butoxide in tert-butanol (0.60ml, 0.0006000 moles) and the product of Example 16[1-(3-bromopentyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane, 0.15 g,0.0005973 moles] were added and the mixture was stirred overnight at 70°C. under argon atmosphere. The solvent was then distilled off underreduced pressure and the residue was dissolved in distilled water (40ml). The pH of the solution was adjusted to 2 with 5% phosphoric acidand the solution was stirred for 15 minutes at room temperature. Next,the pH was readjusted to 12 with 1M sodium hydroxide and the solutionwas stirred for 2 hours while keeping the pH equal to 12 by periodicaddition of 1M sodium hydroxide. The pH was then adjusted to 3 with 5%phosphoric acid and the product was extracted with dichloromethane. Theextract was dried with anhydrous magnesium sulfate and added to ethylether. The precipitate was filtered off and dried under reducedpressure. Yield 1.6 g. NMR (d₆-DMSO): 1.72 ppm (q, CH₂ —CH₂—COO—) 2.24ppm (t, —CH₂—COO—), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone).

Example 17 Formation of mPEG_((5,000 Da))-O—CH[CH₂—O—(CH₂)₃COOH]₂

A solution of mPEG_((5,000 Da))-O—CH(CH₂OH)₂ (2.0 g, 0.0004 moles)(prepared from mPEG_((5,000 Da))-mesylate and 1,3-dibenzyloxy-2-propanolaccording to method described in published U.S. Patent Application US2001/0011115) in toluene (30 ml) was azeotropically dried by distillingoff 15 ml toluene. 1.0M solution of potassium tert-butoxide intert-butanol (2.4 ml, 0.0024 moles) and1-(3-bromopropyl)-4-methyl-2,6,7-trioxabicyclo[2,2,2]octane, 0.60 g,0.0024 moles) (prepared as described in Example 2) were added and themixture was stirred overnight at 70° C. under argon atmosphere. Thesolvent was distilled off under reduced pressure and the residue wasdissolved in distilled water (30 ml). The pH of the solution wasadjusted to 2 with 5% phosphoric acid and the solution was stirred for15 minutes at room temperature. Next, the pH was readjusted to 12 with1M sodium hydroxide and the solution was stirred for 1.5 hours whilemaintaining a pH equal to 12 by the periodic addition of 1M sodiumhydroxide. Thereafter, the pH was adjusted to 3 with 5% phosphoric acidand the product was extracted with dichloromethane. The extract was thendried with anhydrous magnesium sulfate and added to ethyl ether. Theprecipitate was filtered off and dried under reduced pressure. Yield 1.6g. NMR (d₆-DMSO): 1.72 ppm (q, CH₂ —CH₂—COO—) 2.24 ppm (t, —CH₂—COO—),3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone).

Example 18 Formation of mPEG_((5,000 Da))-butanoic Acid

A solution of mPEG_((5,000 Da)) (2.0 g, 0.0004 moles) (NOF Corporation)in toluene (20 ml) was azeotropically dried by distilling off solvent todryness under reduced pressure. The dried material was dissolved in 15ml of anhydrous toluene. 1.0M solution of potassium tert-butoxide intert-butanol (1.2 ml, 0.0012 moles) and trimethyl 4-bromoorthobutyrate(Sigma-Aldrich, 0.25 g, 0.0011 moles) were added and the mixture wasstirred overnight at 70° C. under argon atmosphere. The solvent wasdistilled off under reduced pressure and the residue was dissolved indistilled water (40 ml). The pH of the solution was adjusted to 2 with5% phosphoric acid and the solution was stirred for 15 minutes at roomtemperature. Next the pH was readjusted to 12 with 1M sodium hydroxideand the solution was stirred for 2 hours keeping pH equal 12 by periodicaddition of 1M sodium hydroxide. The pH was adjusted to 3 with 5%phosphoric acid and the product was extracted with dichloromethane. Theextract was dried with anhydrous magnesium sulfate and added to ethylether. The precipitated product was filtered off and dried under reducedpressure. Yield 1.5 g. NMR (d₆-DMSO): 1.72 ppm (q, CH₂ —CH₂—COO—) 2.24ppm (t, —CH₂—COO—), 3.24 ppm (s, —OCH₃), 3.51 ppm (s, PEG backbone).

Example 19 Formation of mPEG-succinimidyl Butanoate

The product of Example 18 (mPEG_((5,000 Da))-butanoic acid) is dissolvedin methylene chloride to form a solution. N-hydroxysuccinimide andN,N-dicyclohexylcarbodiimide is then dissolved in 2 ml of methylenechloride and is added to the solution, which is then stirred overnight.Next, the mixture is filtered and the filtrate is concentrated undervacuum. The product is precipitated by addition of the filtrate toisopropanol and is then collected by filtration and dried under vacuum.The product is represented as follows:

Example 20 Formation of PEGylated Lysozyme

Lysozyme serves as a protein model useful for conjugation reactions.Consequently, other active agent proteins can be substituted forlysozyme in this Example.

Lysozyme solution (4 ml, 3 mg/ml) in 50 ml of pH 6.5 buffer (50 mMsodium phosphate/50 mM NaCl) is added to 20 mg of N-succinimidyl esterof mPEG_((5,000 Da))-butanoic acid (the product of Example 19,mPEG-succinimidyl butanoate). The progress of the reaction is monitoredby capillary electrophoresis over a course of six hours to monitor thereaction. After the six hours, capillary electrophoresis shows evidenceof PEGylated lysozyme.

Example 21 Formation of PEGylated Lysozyme

Lysozyme serves as a protein model useful for conjugation reactions.Consequently, other active agent proteins can be substituted forlysozyme in this Example.

The product of Example 18 (mPEG_((5,000 Da))-butanoic acid) is dissolvedin 20 ml of methylene chloride at room temperature and a solution isformed. The solution is then treated with 1,3-diisopropylcarbodiimide,4-dimethylaminopyridine and lysozyme at 0° C. The reaction solution isthen warmed to room temperature after several hours and kept at roomtemperature for about 16 hours. The reaction mixture is then washed withhydrochloric acid, dried and evaporated to yield the conjugated product.

1. An ortho ester of a branched water-soluble polymer, wherein thebranched water-soluble polymer has two or more polymer arms and theortho ester (a) is attached to each of the two or more polymer arms, and(b) has the following structure,

wherein: (a) is either zero or one; X, when present, is a spacer moiety;(z) is an integer from 1 to 24; R¹, in each occurrence, is independentlyH or an organic radical selected from the group consisting of alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, and substituted aryl; R², in each occurrence, isindependently H or an organic radical selected from the group consistingof alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, and substituted aryl; and each R⁴ is a either(i) an organic radical independently selected from the group consistingof alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, and substituted aryl, or (ii) one or moreatoms that combine with another R⁴ or the remaining R⁴ moieties to forma cyclic ortho ester structure.
 2. The polymer of claim 1, wherein thebranched water-soluble polymer has three polymer arms and the orthoester is attached to each one of the three polymer arms.
 3. The polymerof claim 1, wherein the branched water-soluble polymer has four polymerarms and the ortho ester is attached to each one of the four polymerarms.
 4. The polymer of claim 1, wherein (z) equals three and the orthoester has the following structure:


5. The polymer of claim 4, wherein each occurrence of R¹ and R² is H. 6.The polymer of claim 3, wherein (z) equals three and the ortho ester hasthe following structure:


7. The polymer of claim 6, wherein each occurrence of R¹ and R² is H. 8.The polymer of claim 1, wherein each R⁴ combines with another R⁴ or theremaining R⁴ moieties to faun an ortho ester cyclic structure.
 9. Thepolymer of claim 8, wherein each R⁴ combines with another R⁴ or theremaining R⁴ moieties to form an ortho ester cyclic structure selectedfrom the group consisting of


10. The polymer of claim 6, wherein each R⁴ combines with another R⁴ orthe remaining R⁴ moieties to form an ortho ester cyclic structure. 11.The polymer of claim 10, wherein each R⁴ combines with another R⁴ or theremaining R⁴ moieties to form an ortho ester cyclic structure selectedfrom the group consisting of


12. The polymer of claim 1, wherein the ortho ester has the followingstructure:


13. The polymer of claim 12, wherein (a) is zero and the ortho ester hasthe following structure:


14. The polymer of claim 10, wherein the ortho ester cyclic structurehas the following structure:


15. The polymer of claim 14, wherein (a) is zero and the ortho estercyclic structure has the following structure:


16. The polymer of claim 1, wherein each of the two or more polymer armsis a polymer selected from the group consisting of a poly(alkyleneoxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline,poly(acryloylmorpholine), and poly(oxyethylated polyol).
 17. The polymerof claim 16, wherein each of the two or more polymer arms is apoly(alkylene oxide).
 18. The polymer of claim 17, wherein thepoly(alkylene oxide) is a poly(ethylene glycol).
 19. The polymer ofclaim 18, wherein the poly(ethylene glycol) has a molecular weight offrom about 5,000 daltons to about 100,000 daltons.
 20. The polymer ofclaim 19, wherein the poly(ethylene glycol) has a molecular weight offrom about 5,000 daltons to about 25,000 daltons.
 21. The polymer ofclaim 15, wherein each of the four polymer arms is selected a polymerfrom the group consisting of a poly(alkylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline,poly(acryloylmorpholine), and poly(oxyethylated polyol).
 22. The polymerof claim 21, wherein each of the four more polymer arms is apoly(alkylene oxide).
 23. The polymer of claim 22, wherein thepoly(alkylene oxide) is a poly(ethylene glycol).
 24. The polymer ofclaim 23, wherein the poly(ethylene glycol) has a molecular weight offrom about 5,000 daltons to about 100,000 daltons.
 25. The polymer ofclaim 24, wherein the poly(ethylene glycol) has a molecular weight offrom about 5,000 daltons to about 25,000 daltons.
 26. A polymer selectedfrom the group consisting of

wherein (m) is defined such as to provide —(CH₂CH₂O)— with a molecularweight of from about 1,000 Daltons to about 50,000 Daltons.
 27. Thepolymer of claim 26, wherein the molecular weight is about 30,000Daltons.
 28. The polymer of claim. 26, wherein the molecular weight isabout 40,000 Daltons.